Methods and compositions for the inhibition of trpv4

ABSTRACT

TRPV4 activation increases vascular permeability and can be triggered by both chemical and mechanical cues. This activation of TRPV4 can contribute to a number of pathological conditions, e.g., edema, inflammation, hypertension, and/or hyperalgesia. Described herein are methods and compositions relating to inhibition of mechanically-induced TRPV4 activation, e.g., for the treatment of pulmonary edema, edema, inflammation, hypertension, and/or hyperalgesia.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation under 35 U.S.C. § 120 of co-pendingU.S. application Ser. No. 14/900,265 filed Dec. 21, 2015, which is a 35U.S.C. § 371 National Phase Entry Application of InternationalApplication No. PCT/US2014/043785 filed Jun. 24, 2014, which designatesthe U.S., and which claims benefit under 35 U.S.C. § 119(e) of U.S.Provisional Application No. 61/838,488 filed Jun. 24, 2013, the contentsof which are incorporated herein by reference in their entirety.

GOVERNMENT SUPPORT

This invention was made with federal funding under Grant Nos. CA45548and CA074540 awarded by the National Institutes of Health. The U.S.government has certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 20, 2014, isnamed 701039-077711-PCT_SL.txt and is 48,903 bytes in size.

TECHNICAL FIELD

The technology described herein relates to methods and compositions forthe treatment of diseases involving TRPV4 signaling, e.g. pulmonaryedema.

BACKGROUND

TRPV4 is a transmembrane protein that is expressed in many tissue typesthroughout the body and which has been implicated in a number of diversediseases, ranging from pulmonary edema, to inflammation, tohypertension, to hyperalgesia. It also can regulate vascularpermeability and so may play an important role in development of edema,and provide a mechanism to modulate drug delivery to tumors or acrossthe blood-brain barrier. TRPV4 can be activated both by chemical inputs(e.g. ligand binding) and mechanical inputs (e.g. physical strains andstresses on the cell). While inhibitors of the chemical activation ofTRPV4 are known (e.g. GSK2193874), there is no existing means ofspecifically inhibiting the mechanical activation of TRPV4.

SUMMARY

As described herein, the inventors have identified a domain of CD98which is required for the formation of a CD98-TRPV4-β1integrin complexwhich specifically mediates the mechanically-induced activation ofTRPV4. Accordingly, described herein are compositions and methodsrelating to disrupting this complex and thereby inhibiting mechanicalinduction of TRPV4 activity. TRPV4 plays a role in a number of diseasesand inhibition of TRPV4 therefor has therapeutic use in the treatment ofthose diseases, including, e.g. pulmonary edema, in addition topotentially providing ways to modulate drug delivery across vascularbarriers.

In one aspect, described herein is an isolated polypeptide comprisingthe sequence of SEQ ID NO: 1. In some embodiments, the polypeptide is arecombinant polypeptide. In some embodiments, the polypeptide canfurther comprise a a cell-penetrating agent. In some embodiments, thecell-penetrating agent is selected from the group consisting of TATpolypeptide or a lipid protein delivery reagent, e.g, BIOPORTER™.

In one aspect, described herein is an isolated nucleic acid encoding thepolypeptide described herein. In one aspect, described herein is avector comprising the isolated nucleic acid described herein.

In one aspect, described herein is a pharmaceutical compositioncomprising the polypeptide, the nucleic acid, or the vector describedherein and a pharmaceutically acceptable carrier.

In one aspect, described herein is a method of inhibiting themechanically-dependent activation of TRPV4, the method comprisingadministering a compound that inhibits the interaction of TRPV4 and theN-terminus of CD98 or administering a compound that inhibits theinteraction of the N-terminus of CD98 and an integrin. In one aspect,described herein is a method of treating a disease in a subject, themethod comprising administering a compound that inhibits the interactionof TRPV4 and the N-terminus of CD98 to the subject or administering acompound that inhibits the interaction of the N-terminus of CD98 andintegrin to the subject. In one aspect, described herein is a method oftreating a disease in a subject, the method comprising (a) administeringan antagonist of chemically-dependent TRPV4 signaling to the subject;and (b) administering an antagonist of mechanically-dependent TRPV4signaling to the subject wherein the antagonist ofmechanically-dependent TRPV4 signaling is (i) a compound that inhibitsthe interaction of TRPV4 and the N-terminus of CD98, (ii) a compoundthat inhibits the interaction of the N-terminus of CD98 and integrin,(iii) a compound that binds the extracellular domain of CD98 andinhibits the interaction of TRPV4 and CD98 or (iv) a compound thatmodulates integrin signaling. In some embodiments the disease isselected from the group consisting of pulmonary edema; systemic edema;hypertension; hyperalgesia; inflammation; brachyolmia;spondylometaphyseal dysplasia Kozlowski type; metatropic dysplasia;peripheral neuropathy; asthma; COPD; overactive bladder; incontinence;and acoustic cochlear injury. In some embodiments, the compound which isadministered is a polypeptide, a nucleic acid, a vector, or apharmaceutical composition as described herein. In some embodiments, thecompound is caused to penetrate a cell via electroporation ormagnetoporation.

In one aspect, described herein is a method of identifying an inhibitorof the mechanically-dependent activation of TRPV4, the method comprisingcontacting a complex comprising TRPV4 and CD98, and optionally, integrinwith a candidate agent, measuring the level of complexed TRPV4 and CD98,and optionally, integrin, wherein a decrease in the level of TRPV4complexed with CD98, or the level of integrin complexed with either TRPVor CD98 indicates the candidate can inhibit the mechanically-dependentactivation of TRPV4. In some embodiments, the complex is formed invitro. In some embodiments, the complex is present in a cell. In someembodiments, the level of TRPV4 complexed with CD98 is determined bymeasuring the level of TRPV4 present in focal adhesions in a cell. Insome embodiments, the focal adhesion is detected by the localization ofvinculin.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B demonstrate that β1-integrin, CD98 and TRPV4 co-associate incommon signaling complexes inside HUVE cells. FIG. 1A depicts theresults of an immunoblot that demonstrates that CD98 and β1-integrinco-precipitate from whole cells using anti-TRPV4 antibody. The amount ofeach component in the total cell lysates is given in the left panel. Inthe other two lanes the cell lysates were immunoprecipitated with rabbitIgG as negative control or rabbit anti-TRPV4 antibody. FIG. 1B depictsthe results of immunblots demonstrating that TRPV4 similarlyco-precipitates with either anti-β1-integrin or anti-CD98 antibody. Theamount of TRVP4 is given in the left panel. In the other three lanes,the cell lysates were immunoprecipitated with mouse IgG as a negativecontrol or mouse anti-CD98 antibody or mouse anti-β1-integrin.

FIGS. 2A-2D demonstrate that Ankyrin rich domain of TRPV4 and Highhomology domain of CD98 are important for their binding. FIG. 2A depictsa schematic representation of TRPV4. (PR=proline rich domain; AR=ankyrinrepeat domain,; TM=transmembrane domain) FIG. 2B depicts immunoblotresults demonstrating TRPV4 binding to β1-integrin or CD98. Myc vector,myc-linked full-length TRPV4, myc-linked TRPV4 that lacks proline richdomain (APR), or myc-linked TRPV4 lacking all ankyrin repeat domains(ΔAR1-3) was transfected into HEK293T cells 12 h after plating, and thecells were harvested 24 h later. The right panel gives the Westernblotting for the expression of each transgene in total cell lysates ofrespective transfectants. In the left and middle panels, the lysateswere immunoprecipitated with the anti-β1-integrin or CD98 antibody andthe presence or absence of each moleclule in the immunoprecipitates wasblotted with anti-myc antibody. FIG. 2C depicts a schematicrepresentation of CD98. (HH=high homology domain from Drosophila tomammal; TM=transmembrane domain). FIG. 2D depicts immunoblot resultsdemonstrating CD98 binding to β1-integrin or TRPV4. HA vector, HA-linkedfull-length CD98, HA-linked CD98 that lacks high homology domain (ΔHH)was transfected into HEK293T cells as described in FIG. 2B, cell lysateswere immunoprecipitated with anti-β1-integrin or TRPV4 antibody, and thepresence or absence of each transgene in the precipitates was blottedwith anti-HA antibody.

FIG. 3 demonstrates that CD98 mediates the binding between TRPV4 andβ1-integrin. The cells were transfected with control or CD98 siRNA. Theamount of each component in the total cell lysates is given in the leftpanel. In the right lanses the cell lysates were immunoprecipitates withrabbit anti-TRPV4 (upper) or mouse anti-β1-integrin antibody (lower).

FIG. 4 demonstrates the effects of CD98 high homology domain on thebinding between β1-integrin and TRPV4. 2.5, 5μg HA-linked CD98 highhomology domain or 5μg HA vector was transfected into HEK293T cells, andthe binding assay was performed 24 h later. The bottom panel gives theamount of each transgene in total cell lysates of respectivetransfectants. In the top or the 3rd panel, the lysates wereimmunoprecipitated with an anti-β1-integrin or TRPV4 antibody, and theamount of TRPV4 or β1-integrin in the precipitates was blotted with eachantibodies. The 2^(nd) or the 4^(th) panel gives the amount ofβ1-integrin or TRPV4 in total cell lysates.

FIG. 5 demonstrates that CD98 involves actin rearrangement onmechanotransduction. The graph depicts the percentage of cell orientedat 90±30 degrees (aligned) relative to the direction of applied strainin control and CD98 knockdown HUVE cells; error bars indicate SEM.

FIGS. 6A-6D demonstrate that CD98 is required for mechanical activationof TRPV4. FIG. 6A depicts a graph of the relative change in cytosoliccalcium in response to static stretch (20%, 4 second arrow) inFluo-4-loaded CE cells treated with control or CD98 siRNA. FIG. 6Bdepicts a graph of the average relative increases in cytosolic calciuminduced by mechanical strain in HUVE cells treated with control or CD98siRNA. FIG. 6C depicts a graph of the relative change in cytosoliccalcium in response to 4α-PDD (10 μM) in Fluo-4-loaded CE cells treatedwith control or CD98 siRNA. FIG. 6D depicts a graph of the averagerelative increases in cytosolic calcium induced by chemical strain inHUVE cells treated with control or CD98 siRNA.

FIG. 7 depicts a schematic model of mechanotransduction throughintegrin-CD98-TRPV4 complex. This study proposes that integrintransduces mechanical cues to TRPV4 through CD98. TRPV4 induces calciumsignaling by β1-integrin and also activates integrin signaling.

FIG. 8A depicts a diagram of genetically engineered mutant β1-integrinDNA contruct consisting of the carbonic anhydrase IV (CAIV) enzymeextracellular domain (EC) connected to the transmembrane (TM) domain ofLDL, and the β1-integrin intracellular (IC) domain. FIG. 8B depictsimmunoblot results demonstrating CD98 and TRPV4 bind the same region ofβ1-integrin. β1 construct has full-length intracellular region ofβ1-integrin. β1Δ1 and β1Δ5 are sequential deletion constructs and β1Δ1was replaced the region next to TM domain with alanines. β1 or β1Δ1 β1Δ5was transfected into HEK293T cells 12 h after plating, and the cellswere harvested 24 h later. The bottom panel gives the Western blottingfor the expression of each transgene in total cell lysates of respectivetransfectants. In the upper and middle panels, the lysates wereimmunoprecipitated with the anti TRPV4 or CD98 antibody and the presenceor absence of each molecule in the immunoprecipitates was blotted withanti CAIV antibody.

FIG. 9 depicts the primary sequence alignment of CD98 high homologyregion. In CD98, amino acids 160-201 represents a high homology regionthat is conserves from Drosophila to human (SEQ ID NOS 23-26,respectively, in order of appearance). * indicates identity, : indicateshigh similarity, and . indicates low similarity.

FIG. 10 demonstrates that CD98 mediates the binding between TRPV4 andβ1-integrin. The quantification effects of the experiment depicted inFIG. 3 from n=3 was carried out using ImageJ™ software.

FIG. 11 demonstrates the effects of CD98 high homology domain on thebinding between β1-integrin and TRPV4. The quantification of theexperimental results depicted in FIG. 4 from n=3 was carried out usingImageJ™ software.

FIG. 12 depicts a graph of the percentage of cells oriented 90±30degrees(aligned) relative to the direction of the applied strain in vector orCD98(HH) overexpressing HUVE cells; error bars indicate SEM.

DETAILED DESCRIPTION

Mechanically-dependent activation of TRPV4 is mediated via a complexformed between TRPV4 and CD98 and β1-integrin. As described herein, aparticular domain of CD98, termed the “high homology domain,” isrequired for this complex to form, and the subsequent signaling eventsto occur. Accordingly, compositions and methods to disrupt these TRPV4complexes and the attendant mechanically-activated signaling, e.g. byinhibiting the ability of the high homology domain of CD98 to interactwith TRPV4 and/or β1-integrin, are provided herein.

As described herein, “TRPV4” or “transient receptor potential vanilloid4” refers to a mechanically- and chemically-sensitive calcium channel ofthe TRP channel family. The sequence of TRPV4 for a number of species iswell known in the art, e.g., human TRPV4 (e.g. NCBI Gene ID: 59341;(mRNA: SEQ ID NO: 2, NCBI Ref Seq: NM_021625)(polypeptide: SEQ ID NO: 3,NCBI Ref Seq: NP_067638). TRPV4 can be activated by chemical and/ormechanical input signals. As described herein, “mechanically-dependentactivation of TRPV4” refers to activation of TRPV4 that occurs as aresult of mechanical stimulus, e.g. shear stress, pressure, tension,compression or mechanical strain. Markers of mechanically-dependentTRPV4 activation, and methods of detecting them are known in the art(see, e.g Thodeti et al. Circulation Research 2009 104:1123-1130; whichis incorporated by reference herein in its entirety), and include, butare not limited to calcium influx, PI3K activation, integrinrecruitment, and cytoskeletal remodeling. As described herein,“chemically-dependent activation of TRPV4” refers to activation of TRPV4that occurs as a result of chemical stimulus, e.g. the binding of aligand that stimulates channel opening and activity. TRPV4 activationcan be determined, e.g. by measuring the influx of calcium into a cellcomprising a TRPV4 channel by methods well known in the art.

As used herein, the term “inhibitor” (e.g. inhibitor of TRPV4 activationor of interaction of CD98 and TRPV4 and/or an integrin) refers to anagent which can decrease the targeted activity and/or interaction, e.g.by at least 10% or more, e.g. by 10% or more, 50% or more, 70% or more,80% or more, 90% or more, 95% or more, or 98% or more. Non-limitingexamples of inhibitors of the chemically-dependent activation of TRPV4can include GSK2193874, HC-067047, GSK205, ruthenium red, RN-1734,RN-9893, capsazepine, citral, and the inhibitors described in, e.g.International Patent Publications WO2009/111680, WO2009/146177,WO2009/146182, WO2010/011912, WO2010/011914, and Japanese PatentPublication JP 2009-084209.

As described herein, “integrin” refers to a class of transmembranereceptors that mediate the attachment of a cell to surroundingmaterials, e.g. extracellular matrix (ECM) or other cells, as well astransduce signals relating to the chemical and mechanical status of thesurrounding materials and/or transduce signals from the cell to thesurrounding materials. Integrins function as heterodimers, comprising analpha chain and a beta chain. Mammalian genomes contain eighteen alphasubunits and eight beta subunits. In some embodiments of any of theaspects described herein, an integrin can be a β1-integrin. As describedherein, “β1-integrin” refers to a complete integrin heterodimercomprising a β1 beta chain and any of the eighteen possible alpha chains(e.g. α1-α11, αD, αE, αL, αM, αV, αX or α2B). The sequence of the β1beta chain (i.e. ITGB1) for a number of species is well known in theart, e.g., human ITGB1 (e.g. NCBI Gene ID: 3688; (mRNA: SEQ ID NO: 4,NCBI Ref Seq: NM_002211)(polypeptide: SEQ ID NO: 5, NCBI Ref Seq:NP_002202).

As described herein, “CD98,” “cluster of differentiation 98,” or “LAT1”refers to a heterodimeric membrane transport protein composed of SLC3A2and SLC7A5 which transports certain amino acids across the plasmamembrane (preferentially valine, leucine, isoleucine, tryptophan, andtyrosine). The sequence of SLC3A2 and SLC7A5 for a number of species iswell known in the art, e.g., human SLC3A2 (e.g. NCBI Gene ID: 6520;(mRNA: SEQ ID NO: 6, NCBI Ref Seq: NM_001012662)(polypeptide:SEQ ID NO:7, NCBI Ref Seq: NP_001012680) and human SLC7A5 (e.g. NCBI Gene ID:8140; (mRNA: SEQ ID NO: 8, NCBI Ref Seq: NM_003486)(polypeptide: SEQ IDNO: 9, NCBI Ref Seq: NP 003477). As described herein, the “high homologydomain” of CD98 refers to the domain of CD98 having the sequence of SEQID NO: 1, and/or a domain of CD98 having at least 90%, at least 95%, atleast 98% or greater homology with the sequence of SEQ ID NO: 1.

In one aspect, described herein is an isolated polypeptide comprisingthe sequence of SEQ ID NO: 1. In some embodiments, described herein isan isolated polypeptide consisting of the sequence of SEQ ID NO: 1. Insome embodiments, the isolated polypeptide comprises no more than 300amino acids. In some embodiments, the isolated polypeptide comprises nomore than 200 amino acids. In some embodiments, the isolated polypeptidecomprises no more than 150 amino acids. In some embodiments, theisolated polypeptide comprises no more than 100 amino acids. In someembodiments, the isolated polypeptide comprises no more than 75 aminoacids. The foregoing polypeptides are not naturally-occurringpolypeptides, e.g. SEQ ID NO: 1 naturally occurs in nature only withinthe context of the mature CD98 sequence, as opposed to the engineeredand/or isolated polypeptides described in this paragraph and elsewhereherein. Significantly, these engineered and/or isolated polypeptides(e.g. a polypeptide comprising the sequence of SEQ ID NO: 1 andcomprising no more than 300 amino acids) are demonstrated to possessproperties not possessed by the naturally-occurring mature CD98polypeptide. By way of non-limiting example, the engineered and/orisolated polypeptide can inhibit TRPV4 activation.

An isolated polypeptide as described herein can comprise SEQ ID NO: 1 ora homolog, derivative, variant, conservative substitution variant,deletion mutant, insertion mutant and/or functional fragment thereof. Asused herein, a “functional fragment” of, e.g. SEQ ID NO: 1 is a fragmentor segment of that polypeptide which can inhibit the binding of TRPV4and/or integrin to CD98 at least 10% as strongly as the referencepolypeptide (i.e. SEQ ID NO: 1), e.g. at least 10%, at least 20%, atleast 30%, at least 40%, at least 50%, at least 75%, at least 90%, atleast 100% as strongly, or more strongly. Assays for protein-proteinbinding are well known in the art and include, by way of non-limitingexample, immunoprecipitation or colocalization using at least twoantibodies, one specific for each binding partner. A functional fragmentcan comprise conservative substitutions of the sequences disclosedherein.

Variants of the isolated peptides described herein (e.g. SEQ ID NO: 1)can be obtained by mutations of native nucleotide or amino acidsequences, for example SEQ ID NO: 1 or a nucleotide sequence encoding apeptide comprising SEQ ID NO: 1. A “variant,” as referred to herein, isa polypeptide substantially homologous to the CD98 high homology domaindescribed herein (e.g. SEQ ID NO: 1), but which has an amino acidsequence different from the sequence described herein because of one ora plurality of deletions, insertions or substitutions.

A homolog of an isolated polypeptide as described herein can alsocomprise amino acid sequences that are homologous to the regions of CD98comprised by the isolated polypeptide described herein.

The variant amino acid or DNA sequence preferably is at least 60%, atleast 70%, at least 80%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or more, identical to the sequence from whichit is derived (referred to herein as an “original” sequence). The degreeof homology (percent identity) between an original and a mutant sequencecan be determined, for example, by comparing the two sequences usingfreely available computer programs commonly employed for this purpose onthe world wide web. The variant amino acid or DNA sequence preferably isat least 60%, at least 70%, at least 80%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or more, similar to the sequencefrom which it is derived (referred to herein as an “original” sequence).The degree of similarity (percent similarity) between an original and amutant sequence can be determined, for example, by using a similaritymatrix. Similarity matrices are well known in the art and a number oftools for comparing two sequences using similarity matrices are freelyavailable online, e.g. BLASTp (available on the world wide web atblast.ncbi.nlm.nih.gov).

Alterations of the original amino acid sequence can be accomplished byany of a number of known techniques known to one of skill in the art.Mutations can be introduced, for example, at particular loci bysynthesizing oligonucleotides containing a mutant sequence, flanked byrestriction sites enabling ligation to fragments of the native sequence.Following ligation, the resulting reconstructed sequence encodes ananalog having the desired amino acid insertion, substitution, ordeletion. Alternatively, oligonucleotide-directed site-specificmutagenesis procedures can be employed to provide an altered nucleotidesequence having particular codons altered according to the substitution,deletion, or insertion required. Techniques for making such alterationsinclude those disclosed by Walder et al. (Gene 42:133, 1986); Bauer etal. (Gene 37:73, 1985); Craik (BioTechniques, January 1985, 12-19);Smith et al. (Genetic Engineering: Principles and Methods, Plenum Press,1981); and U.S. Pat. Nos. 4,518,584 and 4,737,462, which are hereinincorporated by reference in their entireties. In some embodiments, anisolated peptide as described herein can be chemically synthesized andmutations can be incorporated as part of the chemical synthesis process.

Variants can comprise conservatively substituted sequences, meaning thatone or more amino acid residues of an original peptide are replaced bydifferent residues, and that the conservatively substituted peptideretains a desired biological activity, i.e., the ability to inhibit thebinding of CD98 to TRPV4 and/or an integrin, that is essentiallyequivalent to that of the original peptide. Examples of conservativesubstitutions include substitutions that do not change the overall orlocal hydrophobic character, substitutions that do not change theoverall or local charge, substitutions by residues of equivalentsidechain size, or substitutions by sidechains with similar reactivegroups.

A given amino acid can be replaced by a residue having similarphysiochemical characteristics, e.g., substituting one aliphatic residuefor another (such as Ile, Val, Leu, or Ala for one another), orsubstitution of one polar residue for another (such as between Lys andArg; Glu and Asp; or Gln and Asn). Other such conservativesubstitutions, e.g., substitutions of entire regions having similarhydrophobicity characteristics or substitutions of residues with similarsidechain volume are well known. Isolated peptides comprisingconservative amino acid substitutions can be tested in any one of theassays described herein to confirm that a desired activity, e.g. theability to inhibit the binding of CD98 to TRPV4 and/or an integrin, isretained, as determined by the assays described elsewhere herein.

Amino acids can be grouped according to similarities in the propertiesof their side chains (in A. L. Lehninger, in Biochemistry, second ed.,pp. 73-75, Worth Publishers, New York (1975)): (1) non-polar: Ala (A),Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2)uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N),Gln (Q); (3) acidic: Asp (D), Glu (E); (4) basic: Lys (K), Arg (R), His(H). Alternatively, naturally occurring residues can be divided intogroups based on common side-chain properties: (1) hydrophobic:Norleucine, Met, Ala, Val, Leu, Ile, Phe, Trp; (2) neutral hydrophilic:Cys, Ser, Thr, Asn, Gln, Ala, Tyr, His, Pro, Gly; (3) acidic: Asp, Glu;(4) basic: His, Lys, Arg; (5) residues that influence chain orientation:Gly, Pro; (6) aromatic: Trp, Tyr, Phe, Pro, His, or hydroxyproline.Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

Particularly preferred conservative substitutions for use in thevariants described herein are as follows: Ala into Gly or into Ser; Arginto Lys; Asn into Gln or into His; Asp into Glu or into Asn; Cys intoSer; Gln into Asn; Glu into Asp; Gly into Ala or into Pro; His into Asnor into Gln; Ile into Leu or into Val; Leu into Ile or into Val; Lysinto Arg, into Gln or into Glu; Met into Leu, into Tyr or into Ile; Pheinto Met, into Leu or into Tyr; Ser into Thr; Thr into Ser; Trp into Tyror into Phe; Tyr into Phe or into Trp; and/or Phe into Val, into Tyr,into Ile or into Leu. In general, conservative substitutions encompassresidue exchanges with those of similar physicochemical properties (i.e.substitution of a hydrophobic residue for another hydrophobic aminoacid).

Any cysteine residue not involved in maintaining the proper conformationof the isolated peptide as described herein can also be substituted,generally with serine, to improve the oxidative stability of themolecule and prevent aberrant crosslinking. Conversely, cysteine bond(s)can be added to the isolated peptide as described herein to improve itsstability or facilitate multimerization.

In some embodiments, the isolated polypeptide further comprises acell-penetrating agent. As used herein, “cell-penetrating agent” refersto an agent, e.g. an amino acid sequence, capable of crossing the lipidbilayer of a cell. Several cell penetrating peptides have beenidentified which can be used as cell-penetrating agents for transportingan isolated polypeptide as described herein across cell membranes. Thesepeptides include, but are not limited to, the homeodomain ofantennapedia, a Drosophila transcription factor (Wang et al., (1995)PNAS USA., 92, 3318-3322); a fragment representing the hydrophobicregion of the signal sequence of Kaposi fibroblast growth factor with orwithout NLS domain (Antopolsky et al. (1999) Bioconj. Chem., 10,598-606); a signal peptide sequence of caiman crocodylus Ig(5) lightchain (Chaloin et al. (1997) Biochem. Biophys. Res. Comm., 243,601-608); a fusion sequence of HIV envelope glycoprotein gp4114, (Morriset al. (1997) Nucleic Acids Res., 25, 2730-2736); a transportanA-achimeric 27-mer consisting of N-terminal fragment of neuropeptidegalanine and membrane interacting wasp venom peptide mastoporan(Lindgren et al., (2000), Bioconjugate Chem., 11, 619-626); a peptidederived from influenza virus hemagglutinin envelop glycoprotein(Bongartz et al., 1994, Nucleic Acids Res., 22, 468 1 4688); RGDpeptide; HIV-1 TAT protein (Frankel and Pabo, (1988) Cell, 55, pp.1189-93). See also, e.g., Morris, M. C. et al., Nature Biotechnol.19:1173-1176 (2001); Dupont, A. J. and Prochiantz, A., CRC Handbook onCell Penetrating Peptides, Langel, Editor, CRC Press, (2002); Chaloin,L. et al., Biochemistry 36(37):11179-87 (1997); and Lundberg, P. andLangel, U., J. Mol. Recognit. 16(5):227-233 (2003); each of which isincorporated herein by reference in its entirety. In some embodiments,the cell-penetrating agent is selected from the group consisting of TATpolypeptide or a lipid protein delivery reagent, e.g, BIOPORTER™.Non-limiting example cell penetration peptide sequences are set forth inTable 1.

TABLE 1 PEPTIDE SEQUENCE SEQ ID NO: HIV-1 TAT (49-57) RKKRRQRRRSEQ ID NO: 10 HIV-1 TAT (48-60) GRKKRRQRRRTPQ SEQ ID NO: 11HIV-1 TAT (47-57) YGRKKRRQRRR SEQ ID NO: 12 Kaposi fibroblastAAV ALL PAV LLA LLA P + SEQ ID NO: 13 growth factor VQR KRQ KLMPSignal peptide MGL GLH LLV LAA ALQ GA SEQ ID NO: 14 sequence of caimancrocodylus Ig(5) light chain HIV envelope GAL FLG FLG AAG STM GA +SEQ ID NO: 15 glycoprotein gp41 PKS KRK 5 (NLS of the SV40) DrosophilaRQI KIW FQN RRM KWK K SEQ ID NO: 16 Antennapedia amide influenza virusGLFEAIAGFIENGWEGMIDGGG SEQ ID NO: 17 hemagglutinin YCenvelop glycoprotein transportan A GWT LNS AGY LLG KIN LKA SEQ ID NO: 18LAA LAK KIL Pre-S-peptide (S)DH QLN PAF SEQ ID NO: 19Somatostatin (tyr-3- (S)FC YWK TCT SEQ ID NO: 20 octreotate) (s)optional Serine for coupling italic = optional D isomer for stability

In another embodiment, the cell penetrating agent can comprise amembrane signal peptide or membrane translocation sequence capable oftranslocating across the cell membrane. A cell penetrating “signalpeptide” or “signal sequence” refers to a sequence of amino acidsgenerally of a length of about 10 to about 50 or more amino acidresidues, many (typically about 55-60%) residues of which arehydrophobic such that they have a hydrophobic, lipid-soluble portion.Generally, a signal peptide is a peptide capable of penetrating throughthe cell membrane to allow the import and/or export of cellularproteins. Signal peptides can be selected from the SIGPEP database (vonHeijne, Protein Sequence Data Analysis 1:4142 (1987); von Heijne andAbrahmsen, L., FEBS Letters 224:439-446 (1989)). Algorithms can alsopredict signal peptide sequences for use in the compositions (see, e.g.,SIGFIND—Signal Peptide Prediction Server version SignalP V2.0b2,Bendtsen et al. “Improved prediction of signal peptides: SignalP 3.0.”J.Mol. Biol., 340:783-795, 2004; Nielsen et al. “Identification ofprokaryotic and eukaryotic signal peptides and prediction of theircleavage sites.” Protein Engineering, 10:1-6, 1997; Bairoch andBoeckmann, “The SWISS-PROT protein sequence data bank: current status”Nucleic Acids Res. 22:3578-3580, 1994.).

In some embodiments of the present invention, the isolated polypeptidecomprises a cell-penetrating agent. In some embodiments, the isolatedpolypeptide can be conjugated to a cell-penetrating agent, e.g. thecell-penetrating agent is not present as fusion protein with, e.g. SEQID NO: 1. For example, the isolated polypeptide may have a cellpenetrating peptide conjugated the polypeptide by chemical bondlinkages, such as linkages by disulfide bonds or by chemical bridges.Peptide sequences of the present invention can also be linked togetherusing non-peptide cross-linkers (Pierce 2003-2004 Applications Handbookand Catalog, Chapter 6) or other scaffolds such as HPMA, polydextran,polysaccharides, ethylene-glycol, poly-ethylene-glycol, glycerol,sugars, and sugar alcohols (e.g., sorbitol, mannitol).

In one aspect, described herein, is an isolated nucleic acid encoding anisolated polypeptide described herein. In one aspect, described herein,is a vector comprising an isolated nucleic acid encoding an isolatedpolypeptide described herein.

Nucleic acid molecules encoding an isolated polypeptide as describedherein are prepared by a variety of methods known in the art. Thesemethods include, but are not limited to, PCR, ligation, and directsynthesis. A nucleic acid sequence encoding a polypeptide as describedherein can be recombined with vector DNA in accordance with conventionaltechniques, including blunt-ended or staggered-ended termini forligation, restriction enzyme digestion to provide appropriate termini,filling in of cohesive ends as appropriate, alkaline phosphatasetreatment to avoid undesirable joining, and ligation with appropriateligases. Techniques for such manipulations are disclosed, e.g., byManiatis et al., Molecular Cloning, Lab. Manual (Cold Spring Harbor Lab.Press, NY, 1982 and 1989), and Ausubel, 1987, 1993, and can be used toconstruct nucleic acid sequences which encode an isolated polypeptide asdescribed herein.

The term “vector” encompasses any genetic element that is capable ofreplication when associated with the proper control elements and thatcan transfer gene sequences to cells. A vector can include, but is notlimited to, a cloning vector, an expression vector, a plasmid, phage,transposon, cosmid, chromosome, virus, virion, etc. These transgenes canbe introduced as a linear construct, a circular plasmid, or a viralvector, which can be an integrating or non-integrating vector. Thetransgene can also be constructed to permit it to be inherited as anextrachromosomal plasmid (Gassmann, et al. , Proc. Natl. Acad. Sci. USA(1995) 92:1292).

In one aspect, the technology described herein relates to an expressionvector comprising a nucleic acid encoding any of the isolatedpolypeptides described herein. Such vectors can be used, e.g. totransform a cell in order to produce the encoded polypeptide. As usedherein, the term “expression vector” refers to a vector that directsexpression of an RNA or polypeptide from sequences linked totranscriptional regulatory sequences on the vector. The sequencesexpressed will often, but not necessarily, be heterologous to the cell.An expression vector may comprise additional elements, for example, theexpression vector may have two replication systems, thus allowing it tobe maintained in two organisms, for example in mammalian cells forexpression and in a prokaryotic host for cloning and amplification. Theterm “expression” refers to the cellular processes involved in producingRNA and proteins and as appropriate, secreting proteins, including whereapplicable, but not limited to, for example, transcription, transcriptprocessing, translation and protein folding, modification andprocessing. “Expression products” include RNA transcribed from a gene,and polypeptides obtained by translation of mRNA transcribed from agene. The term “gene” means the nucleic acid sequence which istranscribed (DNA) to RNA in vitro or in vivo when operably linked toappropriate regulatory sequences. The gene may or may not includeregions preceding and following the coding region, e.g. 5′ untranslated(5′UTR) or “leader” sequences and 3′ UTR or “trailer” sequences, as wellas intervening sequences (introns) between individual coding segments(exons).

By “recombinant vector” is meant a vector that includes a heterologousnucleic acid sequence, or “transgene” that is capable of expression invivo. It should be understood that the vectors described herein can, insome embodiments, be combined with other suitable compositions andtherapies. Vectors useful for the delivery of a sequence encoding anisolated peptide as described herein can include one or more regulatoryelements (e.g., promoter, enhancer, etc.) sufficient for expression ofthe isolated peptide in the desired cell or tissue. The regulatoryelements can be chosen to provide either constitutive orregulated/inducible expression. As used herein, the term “viral vector”refers to a nucleic acid vetor construct that includes at least oneelement of viral origin and has the capacity to be packaged into a viralvector particle. The viral vector can contain the nucleic acid encodingan antibody or antigen-binding portion thereof as described herein inplace of non-essential viral genes. The vector and/or particle may beutilized for the purpose of transferring any nucleic acids into cellseither in vitro or in vivo. Numerous forms of viral vectors are known inthe art.

Examples of vectors useful in delivery of nucleic acids encodingisolated peptides as described herein include plasmid vectors, non-viralplasmid vectors (e.g. see U.S. Pat. Nos. 6,413,942, 6,214,804,5,580,859, 5,589,466, 5,763,270 and 5,693,622, all of which areincorporated herein by reference in their entireties); retroviruses(e.g. see U.S. Pat. No. 5,219,740; Miller and Rosman (1989)BioTechniques 7:980-90; Miller, A. D. (1990) Human Gene Therapy 1:5-14;Scarpa et al. (1991) Virology 180:849-52; Miller et al. , Meth. Enzymol.217:581-599 (1993); Burns et al. (1993) Proc. Natl. Acad. Sci. USA90:8033-37; Boris-Lawrie and Temin (1993) Curr. Opin. Genet. Develop.3:102-09. Boesen et al., Biotherapy 6:291-302 (1994); Clowes et al., J.Clin. Invest. 93:644-651 (1994); Kiem et al., Blood 83:1467-1473 (1994);Salmons and Gunzberg, Human Gene Therapy 4:129-141 (1993); and Grossmanand Wilson, Curr. Opin. in Genetics and Devel. 3:110-114 (1993), thecontents of each of which are herein incorporated by reference in theirentireties); lentiviruses (e.g., see U.S. Pat. Nos. 6,143,520;5,665,557; and 5,981,276, the contents of which are herein incorporatedby reference in their entireties); adenovirus-based expression vectors(e.g., see Haj-Ahmad and Graham (1986) J. Virol. 57:267-74; Bett et al.(1993) J. Virol. 67:5911-21; Mittereder et al. (1994) Human Gene Therapy5:717-29; Seth et al. (1994) J. Virol. 68:933-40; Barr et al. (1994)Gene Therapy 1:51-58; Berkner, K. L. (1988) BioTechniques 6:616-29; andRich et al. (1993) Human Gene Therapy 4:461-76; Wu et al. (2001)Anesthes. 94:1119-32; Parks (2000) Clin. Genet. 58:1-11; Tsai et al.(2000) Curr. Opin. Mol. Ther. 2:515-23; and U.S. Pat. Nos. 6,048,551;6,306,652and 6,306,652, incorporated herein by reference in theirentireties); Adeno-associated viruses (AAV) (e.g. see U.S. Pat. Nos.5,139,941; 5,622,856; 5,139,941; 6,001,650; and 6,004,797, the contentsof each of which are incorporated by reference herein in theirentireties); and avipox vectors (e.g. see WO 91/12882; WO 89/03429; andWO 92/03545; which are incorporated by reference herein in theirentireties).

Useful methods of transfection can include, but are not limited toelectroporation, sonoporation, protoplast fusion, peptoid delivery, ormicroinjection. See, e.g. , Sambrook et al (1989) Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Laboratories, New York, for adiscussion of techniques for transforming cells of interest; andFelgner, P. L. (1990) Advanced Drug Delivery Reviews 5:163-87, for areview of delivery systems useful for gene transfer. Exemplary methodsof delivering DNA using electroporation are described in U.S. Pat. Nos.6,132,419; 6,451,002, 6,418,341, 6,233,483, U.S. Patent Publication No.2002/0146831, and International Publication No. WO/0045823, all of whichare incorporated herein by reference in their entireties.

In some embodiments, the nucleic acid encoding an isolated polypeptideas described herein can be operatively linked to, e.g. a promoter orother transcriptional regulatory sequence. The term “operatively linked”includes having an appropriate start signal (e.g., ATG) in front of thepolynucleotide sequence to be expressed, and maintaining the correctreading frame to permit expression of the polynucleotide sequence underthe control of the expression control sequence, and production of thedesired polypeptide encoded by the polynucleotide sequence. In someexamples, transcription of a nucleic acid modulatory compound is underthe control of a promoter sequence (or other transcriptional regulatorysequence) which controls the expression of the nucleic acid in acell-type in which expression is intended. It will also be understoodthat the modulatory nucleic acid can be under the control oftranscriptional regulatory sequences which are the same or which aredifferent from those sequences which control transcription of thenaturally-occurring form of a protein. In some instances the promotersequence is recognized by the synthetic machinery of the cell, orintroduced synthetic machinery, required for initiating transcription ofa specific gene.

In some embodiments, the vector comprising a nucleic acid encoding anisolated polypeptide as described herein and/or a nucleic acid encodingan isolated polypeptide as described herein, can be present in a cell.The cell can be, e.g. a microbial cell or a mammalian cell. In someembodiments, the cell as described herein is cultured under conditionssuitable for the expression of the isolated polypeptide describedherein. Such conditions can include, but are not limited to, conditionsunder which the cell is capable of growth and/or polypeptide synthesis.Conditions may vary depending upon the species and strain of cellselected. Conditions for the culture of cells, e.g. prokaryotic andmammalian cells, are well known in the art. If the recombinantpolypeptide is operatively linked to an inducible promoter, suchconditions can include the presence of the suitable inducingmolecule(s).

In one aspect, described herein is a pharmaceutical compositioncomprising an isolated polypeptide described herein, a nucleic acidencoding an isolated polypeptide as described herein, and/or a vectorcomprising a nucleic acid encoding an isolated polypeptide as describedherein and a pharmaceutically acceptable carrier.

TRPV4 activity has been implicated in, e.g. breathing motions, ECMstrain, fluid shear stress, pulmonary vascular pressure, barotraumas,serum osmolarity control, nociception, thermal sensing, CNS regulation,bone formation and remodeling, bladder tone, motor and sensoryneuritogenesis, inflammation, adipocyte homeostasis, vascularpermeability and drug delivery. Modulation of TRPV4 therefore has anumber of applications, both therapeutically and as a research tool. Inone aspect, provided herein is a method of inhibiting themechanically-dependent activation of TRPV4, the method comprisingadministering a compound that inhibits the interaction of TRPV4 and theN-terminus of CD98 or administering a compound that inhibits theinteraction of the N-terminus of CD98 and an integrin. The activation ofTRPV4 can be measured in accordance with methods known to one ofordinary skill in the art, e.g. by measuring in the influx of calciuminto the cell, e.g. in the presence of a known stimulus of TRPV4 (e.g.either mechanical or chemical). Methods of measuring calcium flux arewell known in the art, e.g. by measuring the fluorescence of a calciumreporter dye such as Fluo-4 (see, e.g Thodeti et al. CirculationResearch 2009 104:1123-1130; which is incorporated by reference hereinin its entirety)

In one aspect, described herein is a method of treating a disease in asubject, the method comprising administering a compound that inhibitsthe interaction of TRPV4 and the N-terminus of CD98 to the subject oradministering a compound that inhibits the interaction of the N-terminusof CD98 and integrin to the subject. Non-limiting examples of diseasesthat can be treated in accordance with the methods described hereininclude pulmonary edema, systemic edema, hypertension, hyperalgesia,inflammation, brachyolmia, spondylometaphyseal dysplasia Kozlowski type,metatropic dysplasia, peripheral neuropathy, asthma, COPD, overactivebladder, incontinence, and acoustic cochlear injury. Diseases which arecaused, at least in part, by abnormally high levels of TRPV4 signalingare known in the art. See, e.g. Nilius et al. Physiological Reviews 200787:165-217; which is incorporated by reference herein in its entirety.

The compound that inhibits the interaction of TRPV4 and the N-terminusof CD98 and/or inhibits the interaction of the N-terminus of CD98 and anintegrin can be, e.g. an isolated polypeptide as described herein, anucleic acid encoding an isolated polypeptide as described herein, avector comprising a nucleic acid encoding an isolated polypeptide asdescribed herein, or a pharmaceutical composition comprising one or moreof the preceding agents. In some embodiments, the compound can be causedto penetrate a cell via electroporation or magnetoporation.

As the compounds described herein permit specific inhibition ofmechanically-dependent TRPV4 signaling, without impactingchemically-dependent TRPV4 signaling, the combination of an inhibitor ofmechanically-dependent TRPV4 signaling and an inhibitor ofchemically-dependent TRPV4 signaling can be additive and/or synergisticin inhibiting total TRPV4 signaling activity. Accordingly, providedherein is a method of treating a disease in a subject, the methodcomprising administering an antagonist of chemically-dependent TRPV4signaling to the subject and administering an antagonist ofmechanically-dependent TRPV4 signaling to the subject. Inhibitors (e.g.antagonists) of chemically-dependent TRPV4 signaling are describedelsewhere herein. In some embodiments, the antagonist ofmechanically-dependent TRPV4 signaling can be (i) a compound thatinhibits the interaction of TRPV4 and the N-terminus of CD98, (ii) acompound that inhibits the interaction of the N-terminus of CD98 and anintegrin, (iii) a compound that binds the external domain of CD98 andinhibits the interaction of TRPV4 and CD98, and/or (iv) a compound thatmodulates integrin signaling.

As used herein, a compound that binds the extracellular domain of CD98(e.g. amino acids 207-631 of SEQ ID NO: 7 and/or amino acids 76-84,157-170, 221-239, 297-321, 419-432, and/or 479-507 of SEQ ID NO: 9) andinhibits the interaction of TRPV4 and CD98 can be, e.g. an antibody,antibody reagent, peptide, or aptamer. Methods of determining if acompound binds the extracellular domain of CD98, as well as methods fordetecting the interaction of CD98 and TRPV4 are well known in the art,e.g. by immunoprecipitation or immunochemistry, and/or by using labeledreagents and are described elsewhere herein.

As used herein, a compound that inhibits integrin signaling can be acompound that decreases the amount of one or more integrins and/orinhibits integrin activation. A compound that decreases the amount ofone or more integrins, can be, e.g. an inhibitory RNAi that targets oneor more integrin subunits. A compound that inhibits integrin activationcan be, e.g. an agent that binds to integrin, preventingphosphorylation, e.g. an antibody reagent that blocks integrinactivation. Such reagents are known in the art and are commerciallyavailable, e.g. function-blocking anti-β1-integrin (P5D2) available asCat No. sc-13590 (Santa Cruz Biotech, Dallas, Tex.).

In some embodiments, the methods described herein relate to treating asubject having or diagnosed as having a condition described herein, e.g.pulmonary edema, with a method or composition described herein. Subjectshaving, e.g. pulmonary edema can be identified by a physician usingcurrent methods of diagnosing pulmonary edema. Symptoms and/orcomplications of pulmonary edema which characterize these conditions andaid in diagnosis are well known in the art and include but are notlimited to, difficulty breathing, coughing up blood, excessive sweating,anxiety, pale skin, shortness of breath, edema, elevated jugular venouspressure, and hepatomegaly. Tests that may aid in a diagnosis of, e.g.pulmonary edema include, but are not limited to, tests for oxygensaturation in the blood, chest x-ray, echocardiography, blood tests forelectrolytes and renal function, blood count, coagulation test, andtests for levels of B-type natriuretic peptide (BNP). A family historyof pulmonary edema, or exposure to risk factors for pulmonary edema(e.g. heart attack, kidney failure, hypertensive crisis, seizures, headtrauma, or electrocution) can also aid in determining if a subject islikely to have pulmonary edema or in making a diagnosis of pulmonaryedema.

The compositions and methods described herein can be administered to asubject having or diagnosed as having a condition described herein, e.g.pulmonary edema In some embodiments, the methods described hereincomprise administering an effective amount of compositions describedherein, e.g. an isolated polypeptide described herein, to a subject inorder to alleviate a symptom of, e.g. pulmonary edema. As used herein,“alleviating a symptom of” a condition is ameliorating any condition orsymptom associated with the condition. As compared with an equivalentuntreated control, such reduction is by at least 5%, 10%, 20%, 40%, 50%,60%, 80%, 90%, 95%, 99% or more as measured by any standard technique. Avariety of means for administering the compositions described herein tosubjects are known to those of skill in the art. Such methods caninclude, but are not limited to oral, parenteral, intravenous,intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary,cutaneous, injection or topical administration. Administration can belocal or systemic.

The term “effective amount” as used herein refers to the amount of acomposition (e.g. an isolated polypeptide as described herein) needed toalleviate at least one or more symptom of the disease or disorder, andrelates to a sufficient amount of pharmacological composition to providethe desired effect. The term “therapeutically effective amount”therefore refers to an amount of a composition that is sufficient toprovide a particular effect when administered to a typical subject. Aneffective amount as used herein, in various contexts, would also includean amount sufficient to delay the development of a symptom of thedisease, alter the course of a symptom disease (for example but notlimited to, slowing the progression of a symptom of the disease), orreverse a symptom of the disease. Thus, it is not generally practicableto specify an exact “effective amount”. However, for any given case, anappropriate “effective amount” can be determined by one of ordinaryskill in the art using only routine experimentation.

Effective amounts, toxicity, and therapeutic efficacy can be determinedby standard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dosage can vary depending upon the dosage formemployed and the route of administration utilized. The dose ratiobetween toxic and therapeutic effects is the therapeutic index and canbe expressed as the ratio LD50/ED50. Compositions and methods thatexhibit large therapeutic indices are preferred. A therapeuticallyeffective dose can be estimated initially from cell culture assays.Also, a dose can be formulated in animal models to achieve a circulatingplasma concentration range that includes the IC50 (i.e., theconcentration of the active ingredient, which achieves a half-maximalinhibition of symptoms) as determined in cell culture, or in anappropriate animal model. Levels in plasma can be measured, for example,by high performance liquid chromatography. The effects of any particulardosage can be monitored by a suitable bioassay, e.g., assay for fluidleakage in an in vitro model of pulmonary edema as described elsewhereherein, among others. The dosage can be determined by a physician andadjusted, as necessary, to suit observed effects of the treatment.

In some embodiments, the technology described herein relates to apharmaceutical composition comprising, e.g. an isolated polypeptide asdescribed herein, and optionally a pharmaceutically acceptable carrier.Pharmaceutically acceptable carriers and diluents include saline,aqueous buffer solutions, solvents and/or dispersion media. The use ofsuch carriers and diluents is well known in the art. Some non-limitingexamples of materials which can serve as pharmaceutically-acceptablecarriers include: (1) sugars, such as lactose, glucose and sucrose; (2)starches, such as corn starch and potato starch; (3) cellulose, and itsderivatives, such as sodium carboxymethyl cellulose, methylcellulose,ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, suchas magnesium stearate, sodium lauryl sulfate and talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12)esters, such as ethyl oleate and ethyl laurate; (13) agar; (14)buffering agents, such as magnesium hydroxide and aluminum hydroxide;(15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18)Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21)polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents,such as polypeptides and amino acids (23) serum component, such as serumalbumin, HDL and LDL; (22) C₂-C₁₂ alcohols, such as ethanol; and (23)other non-toxic compatible substances employed in pharmaceuticalformulations. Wetting agents, coloring agents, release agents, coatingagents, sweetening agents, flavoring agents, perfuming agents,preservative and antioxidants can also be present in the formulation.The terms such as “excipient”, “carrier”, “pharmaceutically acceptablecarrier” or the like are used interchangeably herein. In someembodiments, the carrier inhibits the degradation of the active agent.

In some embodiments, the pharmaceutical composition can be a parenteraldose form. Since administration of parenteral dosage forms typicallybypasses the patient's natural defenses against contaminants, parenteraldosage forms are preferably sterile or capable of being sterilized priorto administration to a patient. Examples of parenteral dosage formsinclude, but are not limited to, solutions ready for injection, dryproducts ready to be dissolved or suspended in a pharmaceuticallyacceptable vehicle for injection, suspensions ready for injection, andemulsions. In addition, controlled-release parenteral dosage forms canbe prepared for administration of a patient, including, but not limitedto, DUROS®-type dosage forms and dose-dumping.

Suitable vehicles that can be used to provide parenteral dosage formsare well known to those skilled in the art. Examples include, withoutlimitation: sterile water; water for injection USP; saline solution;glucose solution; aqueous vehicles such as but not limited to, sodiumchloride injection, Ringer's injection, dextrose Injection, dextrose andsodium chloride injection, and lactated Ringer's injection;water-miscible vehicles such as, but not limited to, ethyl alcohol,polyethylene glycol, and propylene glycol; and non-aqueous vehicles suchas, but not limited to, corn oil, cottonseed oil, peanut oil, sesameoil, ethyl oleate, isopropyl myristate, and benzyl benzoate. Compoundsthat alter or modify the solubility of a pharmaceutically acceptablesalt can also be incorporated into the parenteral dosage forms of thedisclosure, including conventional and controlled-release parenteraldosage forms.

Pharmaceutical compositions can also be formulated to be suitable fororal administration, for example as discrete dosage forms, such as, butnot limited to, tablets (including without limitation scored or coatedtablets), pills, caplets, capsules, chewable tablets, powder packets,cachets, troches, wafers, aerosol sprays, or liquids, such as but notlimited to, syrups, elixirs, solutions or suspensions in an aqueousliquid, a non-aqueous liquid, an oil-in-water emulsion, or awater-in-oil emulsion. Such compositions contain a predetermined amountof the pharmaceutically acceptable salt of the disclosed compounds, andmay be prepared by methods of pharmacy well known to those skilled inthe art. See generally, Remington: The Science and Practice of Pharmacy,21st Ed., Lippincott, Williams, and Wilkins, Philadelphia Pa. (2005).

Conventional dosage forms generally provide rapid or immediate drugrelease from the formulation. Depending on the pharmacology andpharmacokinetics of the drug, use of conventional dosage forms can leadto wide fluctuations in the concentrations of the drug in a patient'sblood and other tissues. These fluctuations can impact a number ofparameters, such as dose frequency, onset of action, duration ofefficacy, maintenance of therapeutic blood levels, toxicity, sideeffects, and the like. Advantageously, controlled-release formulationscan be used to control a drug's onset of action, duration of action,plasma levels within the therapeutic window, and peak blood levels. Inparticular, controlled- or extended-release dosage forms or formulationscan be used to ensure that the maximum effectiveness of a drug isachieved while minimizing potential adverse effects and safety concerns,which can occur both from under-dosing a drug (i.e., going below theminimum therapeutic levels) as well as exceeding the toxicity level forthe drug. In some embodiments, the composition can be administered in asustained release formulation.

Controlled-release pharmaceutical products have a common goal ofimproving drug therapy over that achieved by their non-controlledrelease counterparts. Ideally, the use of an optimally designedcontrolled-release preparation in medical treatment is characterized bya minimum of drug substance being employed to cure or control thecondition in a minimum amount of time. Advantages of controlled-releaseformulations include: 1) extended activity of the drug; 2) reduceddosage frequency; 3) increased patient compliance; 4) usage of lesstotal drug; 5) reduction in local or systemic side effects; 6)minimization of drug accumulation; 7) reduction in blood levelfluctuations; 8) improvement in efficacy of treatment; 9) reduction ofpotentiation or loss of drug activity; and 10) improvement in speed ofcontrol of diseases or conditions. Kim, Cherng-ju, Controlled ReleaseDosage Form Design, 2 (Technomic Publishing, Lancaster, Pa.: 2000).

Most controlled-release formulations are designed to initially releasean amount of drug (active ingredient) that promptly produces the desiredtherapeutic effect, and gradually and continually release other amountsof drug to maintain this level of therapeutic or prophylactic effectover an extended period of time. In order to maintain this constantlevel of drug in the body, the drug must be released from the dosageform at a rate that will replace the amount of drug being metabolizedand excreted from the body. Controlled-release of an active ingredientcan be stimulated by various conditions including, but not limited to,pH, ionic strength, osmotic pressure, temperature, enzymes, water, andother physiological conditions or compounds.

A variety of known controlled- or extended-release dosage forms,formulations, and devices can be adapted for use with the salts andcompositions of the disclosure. Examples include, but are not limitedto, those described in U.S. Pat. Nos.: 3,845,770; 3,916,899; 3,536,809;3,598,123; 4,008,719; 5,674,533; 5,059,595; 5,591,767; 5,120,548;5,073,543; 5,639,476; 5,354,556; 5,733,566; and 6,365,185 B1; each ofwhich is incorporated herein by reference. These dosage forms can beused to provide slow or controlled-release of one or more activeingredients using, for example, hydroxypropylmethyl cellulose, otherpolymer matrices, gels, permeable membranes, osmotic systems (such asOROS® (Alza Corporation, Mountain View, Calif. USA)), or a combinationthereof to provide the desired release profile in varying proportions.

The methods described herein can further comprise administering a secondagent and/or treatment to the subject, e.g. as part of a combinatorialtherapy. By way of non-limiting example, if a subject is to be treatedfor pain or inflammation according to the methods described herein, thesubject can also be administered a second agent and/or treatment knownto be beneficial for subjects suffering from pain or inflammation.Examples of such agents and/or treatments include, but are not limitedto, non-steroidal anti-inflammatory drugs (NSAIDs—such as aspirin,ibuprofen, or naproxen); corticosteroids, including glucocorticoids(e.g. cortisol, prednisone, prednisolone, methylprednisolone,dexamethasone, betamethasone, triamcinolone, and beclometasone);methotrexate; sulfasalazine; leflunomide; anti-TNF medications;cyclophosphamide; pro-resolving drugs; mycophenolate; or opiates (e.g.endorphins, enkephalins, and dynorphin), steroids, analgesics,barbiturates, oxycodone, morphine, lidocaine, and the like.

In certain embodiments, an effective dose of a composition comprising,e.g. an isolated polypeptide as described herein can be administered toa patient once. In certain embodiments, an effective dose of acomposition can be administered to a patient repeatedly. For systemicadministration, subjects can be administered a therapeutic amount of acomposition, such as, e.g. 0.1 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 2.0 mg/kg,2.5 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 40mg/kg, 50 mg/kg, or more.

In some embodiments, after an initial treatment regimen, the treatmentscan be administered on a less frequent basis. For example, aftertreatment biweekly for three months, treatment can be repeated once permonth, for six months or a year or longer. Treatment according to themethods described herein can reduce levels of a marker or symptom of acondition, e.g. edema in a subject with pulmonary edema by at least 10%,at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80% or at least 90% ormore.

The dosage of a composition as described herein can be determined by aphysician and adjusted, as necessary, to suit observed effects of thetreatment. With respect to duration and frequency of treatment, it istypical for skilled clinicians to monitor subjects in order to determinewhen the treatment is providing therapeutic benefit, and to determinewhether to increase or decrease dosage, increase or decreaseadministration frequency, discontinue treatment, resume treatment, ormake other alterations to the treatment regimen. The dosing schedule canvary from once a week to daily depending on a number of clinicalfactors, such as the subject's sensitivity to the composition. Thedesired dose or amount of activation can be administered at one time ordivided into subdoses, e.g., 2-4 subdoses and administered over a periodof time, e.g., at appropriate intervals through the day or otherappropriate schedule. In some embodiments, administration can bechronic, e.g., one or more doses and/or treatments daily over a periodof weeks or months. Examples of dosing and/or treatment schedules areadministration daily, twice daily, three times daily or four or moretimes daily over a period of 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month,2 months, 3 months, 4 months, 5 months, or 6 months, or more. Acomposition can be administered over a period of time, such as over a 5minute, 10 minute, 15 minute, 20 minute, or 25 minute period.

The dosage ranges for the administration of the compositions describedherein, according to the methods described herein depend upon, forexample, the form of the composition, its potency, and the extent towhich symptoms, markers, or indicators of a condition described hereinare desired to be reduced, for example the percentage reduction desiredfor edema, pain, or inflammation. The dosage should not be so large asto cause adverse side effects. Generally, the dosage will vary with theage, condition, and sex of the patient and can be determined by one ofskill in the art. The dosage can also be adjusted by the individualphysician in the event of any complication.

The efficacy of a composition in, e.g. the treatment of a conditiondescribed herein, or to induce a response as described herein (e.g.modulation of TRPV4 activity) can be determined by the skilledclinician. However, a treatment is considered “effective treatment,” asthe term is used herein, if one or more of the signs or symptoms of acondition described herein are altered in a beneficial manner, otherclinically accepted symptoms are improved, or even ameliorated, or adesired response is induced e.g., by at least 10% following treatmentaccording to the methods described herein. Efficacy can be assessed, forexample, by measuring a marker, indicator, symptom, and/or the incidenceof a condition treated according to the methods described herein or anyother measurable parameter appropriate, e.g. BNP levels in a subjectwith pulmonary edema. Efficacy can also be measured by a failure of anindividual to worsen as assessed by hospitalization, or need for medicalinterventions (i.e., progression of the disease is halted). Methods ofmeasuring these indicators are known to those of skill in the art and/orare described herein. Treatment includes any treatment of a disease inan individual or an animal (some non-limiting examples include a humanor an animal) and includes: (1) inhibiting the disease, e.g., preventinga worsening of symptoms (e.g. pain or inflammation); or (2) relievingthe severity of the disease, e.g., causing regression of symptoms. Aneffective amount for the treatment of a disease means that amount which,when administered to a subject in need thereof, is sufficient to resultin effective treatment as that term is defined herein, for that disease.Efficacy of an agent can be determined by assessing physical indicatorsof a condition or desired response. It is well within the ability of oneskilled in the art to monitor efficacy of administration and/ortreatment by measuring any one of such parameters, or any combination ofparameters. Efficacy can be assessed in animal models of a conditiondescribed herein, for example treatment of pulmonary edema. When usingan experimental animal model, efficacy of treatment is evidenced when astatistically significant change in a marker is observed.

In vitro and animal model assays are provided herein which allow theassessment of a given dose of a composition described herein. By way ofnon-limiting example, the effects of a dose of an isolated polypeptidedescribed herein can be assessed by an in vitro model of pulmonary edemaas described in, e.g. Hamilton et al. Sci Transl Med 2012 4:159; whichis incorporated by reference herein in its entirety. A non-limitingexample of a protocol for such an assay is as follows: IL-2 (1000 U/ml)is perfused through an in vitro model of lung tissue (e.g. lung-on-chip,as described in Hamilton et al. and International Patent PublicationPCT/US2009/050830; each of which is incorporated by reference herein inits entirety). Fluid leakage from the cells can be measured by, e.g.phast contrast microscopy to determine the accumulation of fluid in theair-filled alveolar compartment. The level of fluid leakage can bequantified, e.g. by injecting fluorescein isothiocynate(FITC)-conjugated inulin into the microvascular compartment andmeasuring the fluorescence in fluid subsequently collected in thealveolar compartment.

In one aspect, provided herein is a method of identifying an inhibitorof the mechanically-dependent activation of TRPV4, the method comprisingcontacting a complex comprising TRPV4 and CD98, and optionally, integrinwith a candidate agent, measuring the level of complexed TRPV4 and CD98,and optionally, integrin; wherein a decrease in the level of TRPV4complexed with CD98, or the level of integrin complexed with eitherTRPV4 or CD98 in the presence of the candidate agent indicates thecandidate can inhibit the mechanically-dependent activation of TRPV4. Insome embodiments, the level is decreased if it is lower by astatistically significant amount. In some embodiments, the candidateagent that is screened and identified to inhibit TRPV4mechcanically-dependent activation can decrease the level of, e.g. TRPV4complexed with CD98 by at least 5%, 10%, 20%, 30%, 40%, 50%, 50%, 70%,80%, 90%, 1-fold, 1.1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold,10-fold, 50-fold, 100-fold or more higher relative to an untreatedcontrol.

As used, herein, a “complex” refers to two or more polypeptides (e.g.CD98 and an integrin, TRPV4 and CD98, or CD98, TRPV4, and an integrin)which are bound together. A complex can form when the two or morepolypeptides are present in the same solution and/or cell under suitableconditions, e.g. physiologic pH. In some embodiments, the complex can beformed in vitro, e.g. isolated CD98 and TRPV4 polypeptides, and/or theirsoluble extracellular domains or intracellular domains, can be expressedseparately and then combined under conditions suitable for the formationof a complex in vitro. In some embodiments, the complex can be presentin a cell, e.g. the CD98 and TRPV4 can be expressed in the same cell atdetectable levels. In some embodiments, one or more of the polypeptidescan comprise a detectable label.

In some embodiments the TRPV4, CD98 and/or integrin can be obtained byexpressing the polypeptides in a cell, e.g. using a vector as describedelsewhere herein. In some embodiments, TRPV4, CD98 and/or integrin areexpressed in a eurkaryotic cell. In some embodiments, TRPV4, CD98 and/orintegrin are expressed are synthesized in vitro. The polypeptides can beexpressed and isolated using methods well known to those of ordinaryskill in the art, e.g. as described in Sanbrook et al, MolecularCloning: A Laboratory Manual (3^(rd) Ed) 2001, CSH Press, Cold SpringHarbor, N.Y. Isolated polypeptides can be obtained by similar methods.

In some embodiments, one or more of the polypeptides or agents (e.g. anantibody reagent) described herein can comprise a detectable labeland/or comprise the ability to generate a detectable signal (e.g. bycatalyzing reaction converting a compound to a detectable product).Detectable labels can comprise, for example, a light-absorbing dye, afluorescent dye, or a radioactive label. Detectable labels, methods ofdetecting them, and methods of incorporating them into reagents (e.g.antibodies) are well known in the art.

In some embodiments, detectable labels can include labels that can bedetected by spectroscopic, photochemical, biochemical, immunochemical,electromagnetic, radiochemical, or chemical means, such as fluorescence,chemifluoresence, or chemiluminescence, or any other appropriate means.The detectable labels used in the methods described herein can beprimary labels (where the label comprises a moiety that is directlydetectable or that produces a directly detectable moiety) or secondarylabels (where the detectable label binds to another moiety to produce adetectable signal, e.g., as is common in immunological labeling usingsecondary and tertiary antibodies). The detectable label can be linkedby covalent or non-covalent means to the polypeptide or agent.Alternatively, a detectable label can be linked such as by directlylabeling a molecule that achieves binding to the polypeptide or agentvia a ligand-receptor binding pair arrangement or other such specificrecognition molecules. Detectable labels can include, but are notlimited to radioisotopes, bioluminescent compounds, chromophores,antibodies, chemiluminescent compounds, fluorescent compounds, metalchelates, and enzymes.

In other embodiments, the label is a fluorescent compound. When thefluorescently labeled antibody is exposed to light of the properwavelength, its presence can then be detected due to fluorescence. Insome embodiments, a detectable label can be a fluorescent dye molecule,or fluorophore including, but not limited to fluorescein, phycoerythrin,phycocyanin, o-phthaldehyde, fluorescamine, Cy3™, Cy5™,allophycocyanine, Texas Red, peridenin chlorophyll, cyanine, tandemconjugates such as phycoerythrin-Cy5™, green fluorescent protein,rhodamine, fluorescein isothiocyanate (FITC) and Oregon Green™,rhodamine and derivatives (e.g., Texas red and tetrarhodimineisothiocynate (TRITC)), biotin, phycoerythrin, AMCA, CyDyes™,6-carboxyfhiorescein (commonly known by the abbreviations FAM and F),6-carboxy-2′,4′,7′,4,7-hexachlorofiuorescein (HEX),6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfiuorescein (JOE or J),N,N,N′,N′-tetramethyl-6carboxyrhodamine (TAMRA or T),6-carboxy-X-rhodamine (ROX or R), 5-carboxyrhodamine-6G (R6G5 or G5),6-carboxyrhodamine-6G (R6G6 or G6), and rhodamine 110; cyanine dyes,e.g. Cy3, Cy5 and Cy7 dyes; coumarins, e.g umbelliferone; benzimidedyes, e.g. Hoechst 33258; phenanthridine dyes, e.g. Texas Red; ethidiumdyes; acridine dyes; carbazole dyes; phenoxazine dyes; porphyrin dyes;polymethine dyes, e.g. cyanine dyes such as Cy3, Cy5, etc; BODIPY dyesand quinoline dyes. In some embodiments, a detectable label can be aradiolabel including, but not limited to ³H, ¹²⁵I, ³⁵S, ¹⁴C, ³²P, and³³P. In some embodiments, a detectable label can be an enzyme including,but not limited to horseradish peroxidase and alkaline phosphatase. Anenzymatic label can produce, for example, a chemiluminescent signal, acolor signal, or a fluorescent signal. Enzymes contemplated for use as adetectable label include, but are not limited to, malate dehydrogenase,staphylococcal nuclease, delta-V-steroid isomerase, yeast alcoholdehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphateisomerase, horseradish peroxidase, alkaline phosphatase, asparaginase,glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-VI-phosphate dehydrogenase, glucoamylase andacetylcholinesterase. In some embodiments, a detectable label is achemiluminescent label, including, but not limited to lucigenin,luminol, luciferin, isoluminol, theromatic acridinium ester, imidazole,acridinium salt and oxalate ester. In some embodiments, a detectablelabel can be a spectral colorimetric label including, but not limited tocolloidal gold or colored glass or plastic (e.g., polystyrene,polypropylene, and latex) beads.

In some embodiments, an agent can be detectable by means of a detectabletag, such as c-Myc, HA, VSV-G, HSV, FLAG, V5, HIS, or biotin. Otherdetection systems can also be used, for example, a biotin-streptavidinsystem. In this system, the antibodies immunoreactive (i. e. specificfor) with the biomarker of interest is biotinylated. Quantity ofbiotinylated antibody bound to the biomarker is determined using astreptavidin-peroxidase conjugate and a chromagenic substrate. Suchstreptavidin peroxidase detection kits are commercially available, e. g.from DAKO; Carpinteria, Calif. A label can also be a fluorescenceemitting metal such as ¹⁵²Eu, or others of the lanthanide series. Thesemetals can be attached to the polypeptide or agent using such metalchelating groups as diethylenetriaminepentaacetic acid (DTPA) orethylene diaminetetraacetic acid (EDTA).

In some embodiments, the level of TRPV4 complexed with CD98 isdetermined by measuring the level of TRPV4 present in focal adhesions ina cell. Methods of determining the concentration and/or presence of agiven polypeptide at certain subcellular locations (e.g. focaladhesions) are well known in the art can can include, by way ofnon-limiting example, colocalization of the polypeptide of interest witha molecule known to be present at that location. In some embodiments, afocal adhesion is detected by the localization of vinculin. Antibodyreagents for the detection of vinculin are known in the art and arecommercially available, e.g. Cat No. V9731 (Sigma-Aldrich; St. Louis,Mo.). Antibodies that can be used to detect each of TRPV4, CD98, and agiven integrin (e.g. β1-integrin) are well known in the art andcommercially available (e.g., respectively, Cat Nos. ab39260, 3122-1,and 1798-1; Abcam, Cambridge, Mass.).

As used herein, the terms “candidate compound” or “candidate agent”refer to a compound or agent and/or compositions thereof that are to bescreened for their ability to modulate binding of CD98 to TRPV4 and/orintegrin. In some embodiments, a library of candidate agents can bescreened. Methods for developing small molecule, polymeric and genomebased libraries are described, for example, in Ding, et al. J Am. Chem.Soc. 124: 1594-1596 (2002) and Lynn, et al., J. Am. Chem. Soc. 123:8155-8156 (2001). Commercially available compound libraries can beobtained from, e.g., ArQule (Woburn, Mass.), Panvera (Madison, Wis.),Ryan Scientific (Mt. Pleasant, S.C.), and Enzo Life Sciences (PlymouthMeeting, Pa.).

Generally, compounds can be tested at any concentration that can inhibitcomplex formation relative to a control over an appropriate time period.In some embodiments, compounds are tested at concentration in the rangeof about 0.1 nM to about 1000 mM. In one embodiment, the compound istested in the range of about 0.1 μM to about 20 μM, about 0.1 μM toabout 10 μM, or about 0.1 μM to about 5 μM.

Depending upon the particular embodiment being practiced, the candidatecompounds can be provided free in solution, or may be attached to acarrier, or a solid support, e.g., beads. A number of suitable solidsupports may be employed for immobilization of the candidate compounds.Examples of suitable solid supports include agarose, cellulose, dextran(commercially available as, i.e., Sephadex, Sepharose) carboxymethylcellulose, polystyrene, polyethylene glycol (PEG), filter paper,nitrocellulose, ion exchange resins, plastic films,polyaminemethylvinylether maleic acid copolymer, glass beads, amino acidcopolymer, ethylene-maleic acid copolymer, nylon, silk, etc.Additionally, for the methods described herein, candidate compounds maybe screened individually, or in groups. Group screening is particularlyuseful where hit rates for effective candidate compounds are expected tobe low such that one would not expect more than one positive result fora given group.

For convenience, the meaning of some terms and phrases used in thespecification, examples, and appended claims, are provided below. Unlessstated otherwise, or implicit from context, the following terms andphrases include the meanings provided below. The definitions areprovided to aid in describing particular embodiments, and are notintended to limit the claimed invention, because the scope of theinvention is limited only by the claims. Unless otherwise defined, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs. If there is an apparent discrepancy between the usageof a term in the art and its definition provided herein, the definitionprovided within the specification shall prevail.

For convenience, certain terms employed herein, in the specification,examples and appended claims are collected here.

The terms “decrease”, “reduced”, “reduction”, or “inhibit” are all usedherein to mean a decrease by a statistically significant amount. In someembodiments, “reduce,” “reduction” or “decrease” or “inhibit” typicallymeans a decrease by at least 10% as compared to a reference level (e.g.the absence of a given treatment) and can include, for example, adecrease by at least about 10%, at least about 20%, at least about 25%,at least about 30%, at least about 35%, at least about 40%, at leastabout 45%, at least about 50%, at least about 55%, at least about 60%,at least about 65%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, at least about 99% , or more. As used herein,“reduction” or “inhibition” does not encompass a complete inhibition orreduction as compared to a reference level. “Complete inhibition” is a100% inhibition as compared to a reference level. A decrease can bepreferably down to a level accepted as within the range of normal for anindividual without a given disorder.

The terms “increased”, “increase”, “enhance”, or “activate” are all usedherein to mean an increase by a statically significant amount. In someembodiments, the terms “increased”, “increase”, “enhance”, or “activate”can mean an increase of at least 10% as compared to a reference level,for example an increase of at least about 20%, or at least about 30%, orat least about 40%, or at least about 50%, or at least about 60%, or atleast about 70%, or at least about 80%, or at least about 90% or up toand including a 100% increase or any increase between 10-100% ascompared to a reference level, or at least about a 2-fold, or at leastabout a 3-fold, or at least about a 4-fold, or at least about a 5-foldor at least about a 10-fold increase, or any increase between 2-fold and10-fold or greater as compared to a reference level. In the context of amarker or symptom, a “increase” is a statistically significant increasein such level.

As used herein, a “subject” means a human or animal. Usually the animalis a vertebrate such as a primate, rodent, domestic animal or gameanimal. Primates include chimpanzees, cynomologous monkeys, spidermonkeys, and macaques, e.g., Rhesus. Rodents include mice, rats,woodchucks, ferrets, rabbits and hamsters. Domestic and game animalsinclude cows, horses, pigs, deer, bison, buffalo, feline species, e.g.,domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g.,chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon. Insome embodiments, the subject is a mammal, e.g., a primate, e.g., ahuman. The terms, “individual,” “patient” and “subject” are usedinterchangeably herein.

Preferably, the subject is a mammal. The mammal can be a human,non-human primate, mouse, rat, dog, cat, horse, or cow, but is notlimited to these examples. Mammals other than humans can beadvantageously used as subjects that represent animal models of disease,e.g. pulmonary edema. A subject can be male or female.

The term “ isolated” or “partially purified” as used herein refers to amolecule (e.g. a polypeptide) separated from at least one othercomponent (e.g., a nucleic acid or polypeptide) that is present with themolecule as found in its natural source and/or that would be presentwith the molecule when expressed by a cell, or secreted in the case ofsecreted polypeptides. A chemically synthesized nucleic acid orpolypeptide or one synthesized using in vitro transcription/translationis considered “isolated.”

The term “agent” refers generally to any entity which is normally notpresent or not present at the levels being administered to a cell,tissue or subject. An agent can be selected from a group including butnot limited to: polynucleotides; polypeptides; small molecules; andantibodies or antigen-binding fragments thereof. A polynucleotide can beRNA or DNA, and can be single or double stranded, and can be selectedfrom a group including, for example, nucleic acids and nucleic acidanalogues that encode a polypeptide. A polypeptide can be, but is notlimited to, a naturally-occurring polypeptide, a mutated polypeptide ora fragment thereof that retains the function of interest. Furtherexamples of agents include, but are not limited to a nucleic acidaptamer, peptide-nucleic acid (PNA), locked nucleic acid (LNA), smallorganic or inorganic molecules; saccharide; oligosaccharides;polysaccharides; biological macromolecules, peptidomimetics; nucleicacid analogs and derivatives; extracts made from biological materialssuch as bacteria, plants, fungi, or mammalian cells or tissues andnaturally occurring or synthetic compositions. An agent can be appliedto the media, where it contacts the cell and induces its effects.Alternatively, an agent can be intracellular as a result of introductionof a nucleic acid sequence encoding the agent into the cell and itstranscription resulting in the production of the nucleic acid and/orprotein environmental stimuli within the cell. In some embodiments, theagent is any chemical, entity or moiety, including without limitationsynthetic and naturally-occurring non-proteinaceous entities. In certainembodiments the agent is a small molecule having a chemical moietyselected, for example, from unsubstituted or substituted alkyl,aromatic, or heterocyclyl moieties including macrolides, leptomycins andrelated natural products or analogues thereof. Agents can be known tohave a desired activity and/or property, or can be selected from alibrary of diverse compounds. As used herein, the term “small molecule”can refer to compounds that are “natural product-like,” however, theterm “small molecule” is not limited to “natural product-like”compounds. Rather, a small molecule is typically characterized in thatit contains several carbon—carbon bonds, and has a molecular weight morethan about 50, but less than about 5000 Daltons (5 kD). Preferably thesmall molecule has a molecular weight of less than 3 kD, still morepreferably less than 2 kD, and most preferably less than 1 kD. In somecases it is preferred that a small molecule have a molecular mass equalto or less than 700 Daltons. In some embodiments, an agent can be aninhibitory nucleic acid; an antibody reagent; an antibody; or a smallmolecule.

A subject can be one who has been previously diagnosed with oridentified as suffering from or having a condition in need of treatment(e.g. pulmonary edema) or one or more complications related to such acondition, and optionally, have already undergone treatment for thecondition or the one or more complications related to the condition.Alternatively, a subject can also be one who has not been previouslydiagnosed as having the condition or one or more complications relatedto the condition. For example, a subject can be one who exhibits one ormore risk factors for the condition or one or more complications relatedto the condition or a subject who does not exhibit risk factors.

A “subject in need” of treatment for a particular condition can be asubject having that condition, diagnosed as having that condition, or atrisk of developing that condition.

As used herein, the terms “protein” and “polypeptide” are usedinterchangeably herein to designate a series of amino acid residues,connected to each other by peptide bonds between the alpha-amino andcarboxy groups of adjacent residues. The terms “protein”, and“polypeptide” refer to a polymer of amino acids, including modifiedamino acids (e.g., phosphorylated, glycated, glycosylated, etc.) andamino acid analogs, regardless of its size or function. “Protein” and“polypeptide” are often used in reference to relatively largepolypeptides, whereas the term “peptide” is often used in reference tosmall polypeptides, but usage of these terms in the art overlaps. Theterms “protein” and “polypeptide” are used interchangeably herein whenreferring to a gene product and fragments thereof Thus, exemplarypolypeptides or proteins include gene products, naturally occurringproteins, homologs, orthologs, paralogs, fragments and otherequivalents, variants, fragments, and analogs of the foregoing.

As used herein an “antibody” refers to IgG, IgM, IgA, IgD or IgEmolecules or antigen-specific antibody fragments thereof (including, butnot limited to, a Fab, F(ab′)₂, Fv, disulphide linked Fv, scFv, singledomain antibody, closed conformation multispecific antibody,disulphide-linked scfv, diabody), whether derived from any species thatnaturally produces an antibody, or created by recombinant DNAtechnology; whether isolated from serum, B-cells, hybridomas,transfectomas, yeast or bacteria.

As described herein, an “antigen” is a molecule that is bound by abinding site on an antibody agent. Typically, antigens are bound byantibody ligands and are capable of raising an antibody response invivo. An antigen can be a polypeptide, protein, nucleic acid or othermolecule or portion thereof. The term “antigenic determinant” refers toan epitope on the antigen recognized by an antigen-binding molecule, andmore particularly, by the antigen-binding site of said molecule.

As used herein, the term “antibody reagent” refers to a polypeptide thatincludes at least one immunoglobulin variable domain or immunoglobulinvariable domain sequence and which specifically binds a given antigen.An antibody reagent can comprise an antibody or a polypeptide comprisingan antigen-binding domain of an antibody. In some embodiments, anantibody reagent can comprise a monoclonal antibody or a polypeptidecomprising an antigen-binding domain of a monoclonal antibody. Forexample, an antibody can include a heavy (H) chain variable region(abbreviated herein as VH), and a light (L) chain variable region(abbreviated herein as VL). In another example, an antibody includes twoheavy (H) chain variable regions and two light (L) chain variableregions. The term “antibody reagent” encompasses antigen-bindingfragments of antibodies (e.g., single chain antibodies, Fab and sFabfragments, F(ab′)2, Fd fragments, Fv fragments, scFv, and domainantibodies (dAb) fragments (see, e.g. de Wildt et al., Eur J. Immunol.1996; 26(3):629-39; which is incorporated by reference herein in itsentirety)) as well as complete antibodies. An antibody can have thestructural features of IgA, IgG, IgE, IgD, IgM (as well as subtypes andcombinations thereof). Antibodies can be from any source, includingmouse, rabbit, pig, rat, and primate (human and non-human primate) andprimatized antibodies. Antibodies also include midibodies, humanizedantibodies, chimeric antibodies, and the like.

The VH and VL regions can be further subdivided into regions ofhypervariability, termed “complementarity determining regions” (“CDR”),interspersed with regions that are more conserved, termed “frameworkregions” (“FR”). The extent of the framework region and CDRS has beenprecisely defined (see, Kabat, E. A., et al. (1991) Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242, and Chothia, C.et al. (1987) J. Mol. Biol. 196:901-917; which are incorporated byreference herein in their entireties). Each VH and VL is typicallycomposed of three CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4.

The terms “antigen-binding fragment” or “antigen-binding domain”, whichare used interchangeably herein are used to refer to one or morefragments of a full length antibody that retain the ability tospecifically bind to a target of interest. Examples of binding fragmentsencompassed within the term “antigen-binding fragment” of a full lengthantibody include (i) a Fab fragment, a monovalent fragment consisting ofthe VL, VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalentfragment including two Fab fragments linked by a disulfide bridge at thehinge region; (iii) an Fd fragment consisting of the VH and CH1 domains;(iv) an Fv fragment consisting of the VL and VH domains of a single armof an antibody, (v) a dAb fragment (Ward et al., (1989) Nature341:544-546; which is incorporated by reference herein in its entirety),which consists of a VH or VL domain; and (vi) an isolatedcomplementarity determining region (CDR) that retains specificantigen-binding functionality. As used herein, the term “specificbinding” refers to a chemical interaction between two molecules,compounds, cells and/or particles wherein the first entity binds to thesecond, target entity with greater specificity and affinity than itbinds to a third entity which is a non-target. In some embodiments,specific binding can refer to an affinity of the first entity for thesecond target entity which is at least 10 times, at least 50 times, atleast 100 times, at least 500 times, at least 1000 times or greater thanthe affinity for the third nontarget entity.

Additionally, and as described herein, a recombinant humanized antibodycan be further optimized to decrease potential immunogenicity, whilemaintaining functional activity, for therapy in humans. In this regard,functional activity means a polypeptide capable of displaying one ormore known functional activities associated with a recombinant antibodyor antibody reagent thereof as described herein. Such functionalactivities include, e.g. the ability to bind to the extracellular domainof CD98.

As used herein, the term “nucleic acid” or “nucleic acid sequence”refers to any molecule, preferably a polymeric molecule, incorporatingunits of ribonucleic acid, deoxyribonucleic acid or an analog thereofThe nucleic acid can be either single-stranded or double-stranded. Asingle-stranded nucleic acid can be one nucleic acid strand of adenatured double- stranded DNA. Alternatively, it can be asingle-stranded nucleic acid not derived from any double-stranded DNA.In one aspect, the nucleic acid can be DNA. In another aspect, thenucleic acid can be RNA. Suitable nucleic acid molecules are DNA,including genomic DNA or cDNA. Other suitable nucleic acid molecules areRNA, including mRNA.

In some embodiments, a binding reagent (e.g. an inhibitor that bindstarget described herein) can be an aptamer. Aptamers are short syntheticsingle-stranded oligonucleotides that specifically bind to variousmolecular targets such as small molecules, proteins, nucleic acids, andeven cells and tissues. These small nucleic acid molecules can formsecondary and tertiary structures capable of specifically bindingproteins or other cellular targets, and are essentially a chemicalequivalent of antibodies. Aptamers are highly specific, relatively smallin size, and non-immunogenic. Aptamers are generally selected from abiopanning method known as SELEX (Systematic Evolution of Ligands byExponential enrichment) (Ellington et al. Nature.1990;346(6287):818-822; Tuerk et al., Science. 1990;249(4968):505-510;Ni et al., Curr Med Chem. 2011;18(27):4206-14; which are incorporated byreference herein in their entireties). Methods of generating an apatmerfor any given target are well known in the art. Preclinical studiesusing, e.g. aptamer-siRNA chimeras and aptamer targeted nanoparticletherapeutics have been very successful in mouse models of cancer and HIV(Ni et al., Curr Med Chem. 2011;18(27):4206-14).

Inhibitors of the expression of a given gene can be an inhibitorynucleic acid. In some embodiments, the inhibitory nucleic acid is aninhibitory RNA (iRNA). Double-stranded RNA molecules (dsRNA) have beenshown to block gene expression in a highly conserved regulatorymechanism known as RNA interference (RNAi). The inhibitory nucleic acidsdescribed herein can include an RNA strand (the antisense strand) havinga region which is 30 nucleotides or less in length, i.e., 15-30nucleotides in length, generally 19-24 nucleotides in length, whichregion is substantially complementary to at least part of an mRNAtranscript of, e.g. one or more integrins. The use of these iRNAsenables the targeted degradation of mRNA transcripts, resulting indecreased expression and/or activity of the target (e.g. an integrin).The following detailed description discloses how to make and usecompositions containing iRNAs to inhibit the expression of a target.

As used herein, the term “iRNA” refers to an agent that contains RNA asthat term is defined herein, and which mediates the targeted cleavage ofan RNA transcript via an RNA-induced silencing complex (RISC) pathway.In one embodiment, an iRNA as described herein effects inhibition of theexpression and/or activity of one or more integrins.

In certain embodiments, contacting a cell with the inhibitor (e.g. aniRNA) results in a decrease in the target mRNA level in a cell by atleast about 5%, about 10%, about 20%, about 30%, about 40%, about 50%,about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, up toand including 100% of the target mRNA level found in the cell withoutthe presence of the iRNA.

In one aspect, an RNA interference agent includes a single stranded RNAthat interacts with a target RNA sequence to direct the cleavage of thetarget RNA. Without wishing to be bound by theory, long double strandedRNA introduced into plants and invertebrate cells is broken down intosiRNA by a Type III endonuclease known as Dicer (Sharp et al., GenesDev. 2001, 15:485). Dicer, a ribonuclease-III-like enzyme, processes thedsRNA into 19-23 base pair short interfering RNAs with characteristictwo base 3′ overhangs (Bernstein, et al., (2001) Nature 409:363). ThesiRNAs are then incorporated into an RNA-induced silencing complex(RISC) where one or more helicases unwind the siRNA duplex, enabling thecomplementary antisense strand to guide target recognition (Nykanen, etal., (2001) Cell 107:309). Upon binding to the appropriate target mRNA,one or more endonucleases within the RISC cleaves the target to inducesilencing (Elbashir, et al., (2001) Genes Dev. 15:188). Thus, in oneaspect, an RNA interference agent relates to a double stranded RNA thatpromotes the formation of a RISC complex comprising a single strand ofRNA that guides the complex for cleavage at the target region of atarget transcript to effect silencing of the target gene.

In some embodiments, the iRNA can be a dsRNA. A dsRNA includes two RNAstrands that are sufficiently complementary to hybridize to form aduplex structure under conditions in which the dsRNA will be used. Onestrand of a dsRNA (the antisense strand) includes a region ofcomplementarity that is substantially complementary, and generally fullycomplementary, to a target sequence. The target sequence can be derivedfrom the sequence of an mRNA formed during the expression of the target.The other strand (the sense strand) includes a region that iscomplementary to the antisense strand, such that the two strandshybridize and form a duplex structure when combined under suitableconditions. Generally, the duplex structure is between 15 and 30inclusive, more generally between 18 and 25 inclusive, yet moregenerally between 19 and 24 inclusive, and most generally between 19 and21 base pairs in length, inclusive. Similarly, the region ofcomplementarity to the target sequence is between 15 and 30 inclusive,more generally between 18 and 25 inclusive, yet more generally between19 and 24 inclusive, and most generally between 19 and 21 nucleotides inlength, inclusive. In some embodiments, the dsRNA is between 15 and 20nucleotides in length, inclusive, and in other embodiments, the dsRNA isbetween 25 and 30 nucleotides in length, inclusive. As the ordinarilyskilled person will recognize, the targeted region of an RNA targetedfor cleavage will most often be part of a larger RNA molecule, often anmRNA molecule. Where relevant, a “part” of an mRNA target is acontiguous sequence of an mRNA target of sufficient length to be asubstrate for RNAi-directed cleavage (i.e., cleavage through a RISCpathway). dsRNAs having duplexes as short as 9 base pairs can, undersome circumstances, mediate RNAi-directed RNA cleavage. Most often atarget will be at least 15 nucleotides in length, preferably 15-30nucleotides in length.

One of skill in the art will also recognize that the duplex region is aprimary functional portion of a dsRNA, e.g., a duplex region of 9 to 36,e.g., 15-30 base pairs. Thus, in one embodiment, to the extent that itbecomes processed to a functional duplex of e.g., 15-30 base pairs thattargets a desired RNA for cleavage, an RNA molecule or complex of RNAmolecules having a duplex region greater than 30 base pairs is a dsRNA.Thus, an ordinarily skilled artisan will recognize that in oneembodiment, then, an miRNA is a dsRNA. In another embodiment, a dsRNA isnot a naturally occurring miRNA. In another embodiment, an iRNA agentuseful to target expression of a target is not generated in the targetcell by cleavage of a larger dsRNA.

In yet another embodiment, the RNA of an iRNA, e.g., a dsRNA, ischemically modified to enhance stability or other beneficialcharacteristics. The nucleic acids featured in the invention may besynthesized and/or modified by methods well established in the art, suchas those described in “Current protocols in nucleic acid chemistry,”Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y.,USA, which is hereby incorporated herein by reference. Modificationsinclude, for example, (a) end modifications, e.g., 5′ end modifications(phosphorylation, conjugation, inverted linkages, etc.) 3′ endmodifications (conjugation, DNA nucleotides, inverted linkages, etc.),(b) base modifications, e.g., replacement with stabilizing bases,destabilizing bases, or bases that base pair with an expanded repertoireof partners, removal of bases (abasic nucleotides), or conjugated bases,(c) sugar modifications (e.g., at the 2′ position or 4′ position) orreplacement of the sugar, as well as (d) backbone modifications,including modification or replacement of the phosphodiester linkages.Specific examples of RNA compounds useful in the embodiments describedherein include, but are not limited to RNAs containing modifiedbackbones or no natural internucleoside linkages. RNAs having modifiedbackbones include, among others, those that do not have a phosphorusatom in the backbone. For the purposes of this specification, and assometimes referenced in the art, modified RNAs that do not have aphosphorus atom in their internucleoside backbone can also be consideredto be oligonucleosides. In particular embodiments, the modified RNA willhave a phosphorus atom in its internucleoside backbone.

Modified RNA backbones can include, for example, phosphorothioates,chiral phosphorothioates, phosphorodithioates, phosphotriesters,aminoalkylphosphotriesters, methyl and other alkyl phosphonatesincluding 3′-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs ofthese, and those) having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Varioussalts, mixed salts and free acid forms are also included.

Representative U.S. patents that teach the preparation of the abovephosphorus-containing linkages include, but are not limited to, U.S.Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799;5,587,361; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170;6,172,209; 6, 239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423;6,531,590; 6,534,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294;6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and US PatRE39464, each of which is herein incorporated by reference

Modified RNA backbones that do not include a phosphorus atom thereinhave backbones that are formed by short chain alkyl or cycloalkylinternucleoside linkages, mixed heteroatoms and alkyl or cycloalkylinternucleoside linkages, or one or more short chain heteroatomic orheterocyclic internucleoside linkages. These include those havingmorpholino linkages (formed in part from the sugar portion of anucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH₂ component parts.

Representative U.S. patents that teach the preparation of the aboveoligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046;5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and,5,677,439, each of which is herein incorporated by reference.

In other RNA mimetics suitable or contemplated for use in iRNAs, boththe sugar and the internucleoside linkage, i.e., the backbone, of thenucleotide units are replaced with novel groups. The base units aremaintained for hybridization with an appropriate nucleic acid targetcompound. One such oligomeric compound, an RNA mimetic that has beenshown to have excellent hybridization properties, is referred to as apeptide nucleic acid (PNA). In PNA compounds, the sugar backbone of anRNA is replaced with an amide containing backbone, in particular anaminoethylglycine backbone. The nucleobases are retained and are bounddirectly or indirectly to aza nitrogen atoms of the amide portion of thebackbone. Representative U.S. patents that teach the preparation of PNAcompounds include, but are not limited to, U.S. Pat. Nos. 5,539,082;5,714,331; and 5,719,262, each of which is herein incorporated byreference. Further teaching of PNA compounds can be found, for example,in Nielsen et al., Science, 1991, 254, 1497-1500.

Some embodiments featured in the invention include RNAs withphosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH₂—NH—CH₂—, —CH₂—N(CH₃)—O—CH₂—[known as amethylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —N(CH₃)—CH₂—CH₂—[wherein the nativephosphodiester backbone is represented as —O—P—O—CH₂—] of theabove-referenced U.S. Pat. No. 5,489,677, and the amide backbones of theabove-referenced U.S. Pat. No. 5,602,240. In some embodiments, the RNAsfeatured herein have morpholino backbone structures of theabove-referenced U.S. Pat. No. 5,034,506.

Modified RNAs can also contain one or more substituted sugar moieties.The iRNAs, e.g., dsRNAs, featured herein can include one of thefollowing at the 2′ position: OH; F; O—, S—, or N-alkyl; O—, S—, orN-alkenyl; O—, S— or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl,alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀ alkylor C₂ to C₁₀ alkenyl and alkynyl. Exemplary suitable modificationsinclude O[(CH₂)_(n)O] _(m)CH₃, O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂,O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where nand m are from 1 to about 10. In other embodiments, dsRNAs include oneof the following at the 2′ position: C₁ to C₁₀ lower alkyl, substitutedlower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN,Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂,heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino,substituted silyl, an RNA cleaving group, a reporter group, anintercalator, a group for improving the pharmacokinetic properties of aniRNA, or a group for improving the pharmacodynamic properties of aniRNA, and other substituents having similar properties. In someembodiments, the modification includes a 2′-methoxyethoxy(2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martinet al., Helv. Chim. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxygroup. Another exemplary modification is 2′-dimethylaminooxyethoxy,i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described inexamples herein below, and 2′-dimethylaminoethoxyethoxy (also known inthe art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e2′-O—CH₂—O—CH₂—N(CH₂)₂, also described in examples herein below.

Other modifications include 2′-methoxy (2′-OCH₃), 2′-aminopropoxy(2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similar modifications can alsobe made at other positions on the RNA of an iRNA, particularly the 3′position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linkeddsRNAs and the 5′ position of 5′ terminal nucleotide. iRNAs may alsohave sugar mimetics such as cyclobutyl moieties in place of thepentofuranosyl sugar. Representative U.S. patents that teach thepreparation of such modified sugar structures include, but are notlimited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044;5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811;5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873;5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which arecommonly owned with the instant application, and each of which is hereinincorporated by reference.

An iRNA can also include nucleobase (often referred to in the art simplyas “base”) modifications or substitutions. As used herein, “unmodified”or “natural” nucleobases include the purine bases adenine (A) andguanine (G), and the pyrimidine bases thymine (T), cytosine (C) anduracil (U). Modified nucleobases include other synthetic and naturalnucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine,xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil,cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo,8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8-substitutedadenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyland other 5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-daazaadenine and 3-deazaguanine and 3-deazaadenine. Furthernucleobases include those disclosed in U.S. Pat. No. 3,687,808, thosedisclosed in Modified Nucleosides in Biochemistry, Biotechnology andMedicine, Herdewijn, P. ed. Wiley-VCH, 2008; those disclosed in TheConcise Encyclopedia Of Polymer Science And Engineering, pages 858-859,Kroschwitz, J. L, ed. John Wiley & Sons, 1990, these disclosed byEnglisch et al., Angewandte Chemie, International Edition, 1991, 30,613, and those disclosed by Sanghvi, Y S., Chapter 15, dsRNA Researchand Applications, pages 289-302, Crooke, S. T. and Lebleu, B., Ed., CRCPress, 1993. Certain of these nucleobases are particularly useful forincreasing the binding affinity of the oligomeric compounds featured inthe invention. These include 5-substituted pyrimidines, 6-azapyrimidinesand N-2, N-6 and 0-6 substituted purines, including2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5-methylcytosine substitutions have been shown to increase nucleic acidduplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. andLebleu, B., Eds., dsRNA Research and Applications, CRC Press, BocaRaton, 1993, pp. 276-278) and are exemplary base substitutions, evenmore particularly when combined with 2′-O-methoxyethyl sugarmodifications.

Representative U.S. patents that teach the preparation of certain of theabove noted modified nucleobases as well as other modified nucleobasesinclude, but are not limited to, the above noted U.S. Pat. No.3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,30; 5,134,066;5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908;5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091;5,614,617; 5,681,941; 6,015,886; 6,147,200; 6,166,197; 6,222,025;6,235,887; 6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610;7,427,672; and 7,495,088, each of which is herein incorporated byreference, and U.S. Pat. No. 5,750,692, also herein incorporated byreference.

The RNA of an iRNA can also be modified to include one or more lockednucleic acids (LNA). A locked nucleic acid is a nucleotide having amodified ribose moiety in which the ribose moiety comprises an extrabridge connecting the 2′ and 4′ carbons. This structure effectively“locks” the ribose in the 3′-endo structural conformation. The additionof locked nucleic acids to siRNAs has been shown to increase siRNAstability in serum, and to reduce off-target effects (Elmen, J. et al.,(2005) Nucleic Acids Research 33(1):439-447; Mook, OR. et al., (2007)Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic AcidsResearch 31(12):3185-3193). Representative U.S. Patents that teach thepreparation of locked nucleic acid nucleotides include, but are notlimited to, the following: U.S. Pat. Nos. 6,268,490; 6,670,461;6,794,499; 6,998,484; 7,053,207; 7,084,125; and 7,399,845, each of whichis herein incorporated by reference in its entirety.

Another modification of the RNA of an iRNA featured in the inventioninvolves chemically linking to the RNA one or more ligands, moieties orconjugates that enhance the activity, cellular distribution,pharmacokinetic properties, or cellular uptake of the iRNA. Suchmoieties include but are not limited to lipid moieties such as acholesterol moiety (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989,86: 6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let.,1994, 4:1053-1060), a thioether, e.g., beryl-S-tritylthiol (Manoharan etal., Ann. N.Y. Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg.Med. Chem. Let., 1993, 3:2765-2770), a thiocholesterol (Oberhauser etal., Nucl. Acids Res., 1992, 20:533-538), an aliphatic chain, e.g.,dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991,10:1111-1118; Kabanov et al., FEBS Lett., 1990, 259:327-330; Svinarchuket al., Biochimie, 1993, 75:49-54), a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethyl-ammonium1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res.,1990, 18:3777-3783), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995,36:3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264:229-237), or an octadecylamine orhexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277:923-937).

As used herein, the terms “treat,” “treatment,” “treating,” or“amelioration” refer to therapeutic treatments, wherein the object is toreverse, alleviate, ameliorate, inhibit, slow down or stop theprogression or severity of a condition associated with a disease ordisorder, e.g. pulmonary edema. The term “treating” includes reducing oralleviating at least one adverse effect or symptom of a condition,disease or disorder, e.g. pulmonary edema. Treatment is generally“effective” if one or more symptoms or clinical markers are reduced.Alternatively, treatment is “effective” if the progression of a diseaseis reduced or halted. That is, “treatment” includes not just theimprovement of symptoms or markers, but also a cessation of, or at leastslowing of, progress or worsening of symptoms compared to what would beexpected in the absence of treatment. Beneficial or desired clinicalresults include, but are not limited to, alleviation of one or moresymptom(s), diminishment of extent of disease, stabilized (i.e., notworsening) state of disease, delay or slowing of disease progression,amelioration or palliation of the disease state, remission (whetherpartial or total), and/or decreased mortality, whether detectable orundetectable. The term “treatment” of a disease also includes providingrelief from the symptoms or side-effects of the disease (includingpalliative treatment).

As used herein, the term “pharmaceutical composition” refers to theactive agent in combination with a pharmaceutically acceptable carriere.g. a carrier commonly used in the pharmaceutical industry. The phrase“pharmaceutically acceptable” is employed herein to refer to thosecompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

As used herein, the term “administering,” refers to the placement of acompound as disclosed herein into a subject by a method or route whichresults in at least partial delivery of the agent at a desired site.Pharmaceutical compositions comprising the compounds disclosed hereincan be administered by any appropriate route which results in aneffective treatment in the subject.

The term “statistically significant” or “significantly” refers tostatistical significance and generally means a two standard deviation(2SD) or greater difference.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used in connection with percentages canmean ±1%.

As used herein the term “comprising” or “comprises” is used in referenceto compositions, methods, and respective component(s) thereof, that areessential to the method or composition, yet open to the inclusion ofunspecified elements, whether essential or not.

The term “consisting of” refers to compositions, methods, and respectivecomponents thereof as described herein, which are exclusive of anyelement not recited in that description of the embodiment.

As used herein the term “consisting essentially of” refers to thoseelements required for a given embodiment. The term permits the presenceof elements that do not materially affect the basic and novel orfunctional characteristic(s) of that embodiment.

The singular terms “a,” “an,” and “the” include plural referents unlesscontext clearly indicates otherwise. Similarly, the word “or” isintended to include “and” unless the context clearly indicatesotherwise. Although methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of thisdisclosure, suitable methods and materials are described below. Theabbreviation, “e.g.” is derived from the Latin exempli gratia, and isused herein to indicate a non-limiting example. Thus, the abbreviation“e.g.” is synonymous with the term “for example.”

Definitions of common terms in cell biology and molecular biology can befound in “The Merck Manual of Diagnosis and Therapy”, 19th Edition,published by Merck Research Laboratories, 2006 (ISBN 0-911910-19-0);Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Biology,published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); BenjaminLewin, Genes X, published by Jones & Bartlett Publishing, 2009 (ISBN-10:0763766321); Kendrew et al. (eds.), Molecular Biology and Biotechnology:a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995(ISBN 1-56081-569-8) and Current Protocols in Protein Sciences 2009,Wiley Intersciences, Coligan et al., eds.

Unless otherwise stated, the present invention was performed usingstandard procedures, as described, for example in Sambrook et al.,Molecular Cloning: A Laboratory Manual (3 ed.), Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., USA (2001); Davis et al.,Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc.,New York, USA (1995); or Methods in Enzymology: Guide to MolecularCloning Techniques Vol.152, S. L. Berger and A. R. Kimmel Eds., AcademicPress Inc., San Diego, USA (1987); Current Protocols in Protein Science(CPPS) (John E. Coligan, et. al., ed., John Wiley and Sons, Inc.),Current Protocols in Cell Biology (CPCB) (Juan S. Bonifacino et. al.ed., John Wiley and Sons, Inc.), and Culture of Animal Cells: A Manualof Basic Technique by R. Ian Freshney, Publisher: Wiley-Liss; 5thedition (2005), Animal Cell Culture Methods (Methods in Cell Biology,Vol. 57, Jennie P. Mather and David Barnes editors, Academic Press, 1stedition, 1998) which are all incorporated by reference herein in theirentireties.

Other terms are defined herein within the description of the variousaspects of the invention.

All patents and other publications; including literature references,issued patents, published patent applications, and co-pending patentapplications; cited throughout this application are expresslyincorporated herein by reference for the purpose of describing anddisclosing, for example, the methodologies described in suchpublications that might be used in connection with the technologydescribed herein. These publications are provided solely for theirdisclosure prior to the filing date of the present application. Nothingin this regard should be construed as an admission that the inventorsare not entitled to antedate such disclosure by virtue of priorinvention or for any other reason. All statements as to the date orrepresentation as to the contents of these documents is based on theinformation available to the applicants and does not constitute anyadmission as to the correctness of the dates or contents of thesedocuments.

The description of embodiments of the disclosure is not intended to beexhaustive or to limit the disclosure to the precise form disclosed.While specific embodiments of, and examples for, the disclosure aredescribed herein for illustrative purposes, various equivalentmodifications are possible within the scope of the disclosure, as thoseskilled in the relevant art will recognize. For example, while methodsteps or functions are presented in a given order, alternativeembodiments may perform functions in a different order, or functions maybe performed substantially concurrently. The teachings of the disclosureprovided herein can be applied to other procedures or methods asappropriate. The various embodiments described herein can be combined toprovide further embodiments. Aspects of the disclosure can be modified,if necessary, to employ the compositions, functions and concepts of theabove references and application to provide yet further embodiments ofthe disclosure. Moreover, due to biological functional equivalencyconsiderations, some changes can be made in protein structure withoutaffecting the biological or chemical action in kind or amount. These andother changes can be made to the disclosure in light of the detaileddescription. All such modifications are intended to be included withinthe scope of the appended claims.

Specific elements of any of the foregoing embodiments can be combined orsubstituted for elements in other embodiments. Furthermore, whileadvantages associated with certain embodiments of the disclosure havebeen described in the context of these embodiments, other embodimentsmay also exhibit such advantages, and not all embodiments neednecessarily exhibit such advantages to fall within the scope of thedisclosure.

The technology described herein is further illustrated by the followingexamples which in no way should be construed as being further limiting.

Some embodiments of the technology described herein can be definedaccording to any of the following numbered paragraphs:

-   -   1. An isolated polypeptide comprising the sequence of SEQ ID NO:        1    -   2. An isolated polypeptide of paragraph 1, further comprising a        cell-penetrating agent.    -   3. The polypeptide of paragraph 2, wherein the cell-penetrating        agent is selected from the group consisting of:        -   TAT polypeptide or a lipid protein delivery reagent, e.g,            BIOPORTER™.    -   4. An isolated nucleic acid encoding the polypeptide of any of        paragraphs 1-3.    -   5. The nucleic acid of paragraph 4, wherein the nucleic acid is        a cDNA.    -   6. A vector comprising the isolated nucleic acid of any of        paragraphs 4-5.    -   7. A pharmaceutical composition comprising the polypeptide of        any of paragraphs 1-3, the nucleic acid of any of paragraphs        4-5, or the vector of paragraph 6 and a pharmaceutically        acceptable carrier.    -   8. A method of inhibiting the mechanically-dependent activation        of TRPV4, the method comprising:        -   administering a compound that inhibits the interaction of            TRPV4 and the N-terminus of CD98; or        -   administering a compound that inhibits the interaction of            the N-terminus of CD98 and an integrin.    -   9. A method of treating a disease in a subject, the method        comprising:        -   administering a compound that inhibits the interaction of            TRPV4 and the N-terminus of CD98 to the subject; or        -   administering a compound that inhibits the interaction of            the N-terminus of CD98 and integrin to the subject.    -   10. A method of treating a disease in a subject, the method        comprising;        -   (a) administering an antagonist of chemically-dependent            TRPV4 signaling to the subject; and        -   (b) administering an antagonist of mechanically-dependent            TRPV4 signaling to the subject;        -   wherein the antagonist of mechanically-dependent TRPV4            signaling is:        -   (i) a compound that inhibits the interaction of TRPV4 and            the N-terminus of CD98;        -   (ii) a compound that inhibits the interaction of the            N-terminus of CD98 and integrin;        -   (iii) a compound that binds the extracellular domain of CD98            and inhibits the interaction of TRPV4 and CD98;        -   (iv) a compound that modulates integrin signaling.    -   11. The method of paragraph 10, wherein the disease is selected        from the group consisting of:        -   pulmonary edema; systemic edema; hypertension; hyperalgesia;            inflammation; brachyolmia; spondylometaphyseal dysplasia            Kozlowski type; metatropic dysplasia; peripheral neuropathy;            asthma; COPD; overactive bladder; incontinence; and acoustic            cochlear injury.    -   12. The method of any of paragraphs 8-11, wherein the compound        which is administered is a polypeptide of any of paragraphs 1-3,        the nucleic acid of any of paragraphs 4-5, the vector of        paragraph 6, or the pharmaceutical composition of paragraph 7.    -   13. The method of paragraph 12, wherein the compound is caused        to penetrate a cell via electroporation or magnetoporation.    -   14. A method of identifying an inhibitor of the        mechanically-dependent activation of TRPV4, the method        comprising:        -   contacting a complex comprising TRPV4 and CD98, and            optionally, integrin with a candidate agent;        -   measuring the level of complexed TRPV4 and CD98, and            optionally, integrin; wherein a decrease in the level of            TRPV4 complexed with CD98, or the level of integrin            complexed with either TRPV or CD98 indicates the candidate            can inhibit the mechanically-dependent activation of TRPV4.    -   15. The method of paragraph 14, wherein the complex is formed in        vitro.    -   16. The method of paragraph 14, wherein the complex is present        in a cell.    -   17. The method of paragraph 16, wherein the level of TRPV4        complexed with CD98 is determined by measuring the level of        TRPV4 present in focal adhesions in a cell.    -   18. The method of paragraph 17, wherein the focal adhesion is        detected by the localization of vinculin.

EXAMPLES Example 1 CD98 Mediates Mechanical Signal from β1-Integrin toTRPV4

The conversion of physical force into biochemical information isfundamental to development and physiology. One of the most rapidmechanical events involves integrin-dependent activation of thestress-activated (SA) membrane ion channel Transient receptor potentialvanilloid 4 (TRPV4). However, it is still unclear the molecularmechanism by which integrins mediate these “early-immediate” mechanicalsignaling responses that activate TRPV4. It is reported herein that CD98mediates mechanical activation of the TRPV4 membrane ion channel byphysical forces applied to β1-integrins. As demonstrated herein, TRPV4associates with β1-integrin and CD98 and localizes at focal adhesion inHUVE cells. Although the formation of focal adhesion occurs, TRPV4 can'tlocalize at focal adhesion in CD98 siRNA-treated cells. Moreover, TRPV4can't bind to β1-integrin in CD98 siRNA-treated cells. In addition, itis demonstrated herein that actin rearrangement and calcium influx areinhibited in CD98 siRNA-treated cells. Together these findingsdemonstrate that CD98 plays a role in mechanotransduction by mediatingthe binding between β1-integrin and TRPV4 at focal adhesion.Additionally, the calcium influx of TRPV4 activation by chemicaltreatment was not inhibited in CD98 siRNA-treated cells. Without wishingto be bound by theory, the pathway of TRPV4 activation may be differentbetween chemical and mechanical cues.

Introduction

Essentially all organisms from bacteria to humans are mechanosensitive.Cellular mechanotransduction—the mechanism by which cells sensemechanical forces and convert them into changes in cellularbiochemistry—is critical for control of the growth and development ofall tissues, and deregulation of this process contributes to theetiology of numerous diseases (1). For example, pressure and shearstress from pumping blood influence the morphology and pathology of theheart and vasculature in the vascular system. Bone is shaped by forcesfrom gravity and muscle contraction. Hearing and touch are based onneural responses to pressure. Inflation and deflation of the lungsregulate their physiology. Coordinated growth of tissues is guided bymechanical forces, and failure of these mechanisms contributes tocancer. Therefore, to elucidate the force transmission pathway in thecell body lead to benefits for developing new drugs and treatments forthese diseases.

It is reported that mechanotransduction brings about important cellularchanges in shape, motility, cytoskeletal remodeling, focal adhesionreorganization and gene expression (2). However, the mechanism by whichcells transmit mechanical stress throughout the cytoplasm and thecytoskeleton and how such signals are sensed and converted intobiochemical signals is still not understood.

An important group of adhesive transmembrane receptors that mechanicallylink the Extracellular matrix (ECM) with internal cytoskeleton areintegrins. Transmembrane integrin receptors that support ECM adhesionand physically couple ECM to the cytoskeleton mediatemechanotransduction (3). Stress activated (SA) channels representanother class of mechanoreceptors that support the conversion ofmechanical force signals applied to the cell surface into transmembraneion gradients (4). TRPV4 belongs to the Transient Receptor Potential(TRP) superfamily, which consists of at least 33 channel subunit genesdivided into 6 sub-families. These proteins are a non-selectivecalcium-permeable cation channels. They are activated and regulated by avariety of stimuli and are expressed widely. TRPC1, TRPC6, TRPA1, TRPM3,TRPM7 TRPV2 and TRPV4 are reported as mechanosensors among the TRPfamily (5).

TRPV4 was previously identified as the SA channel responsible foractivation of microvascular endothelial cells when mechanical forces areapplied to β1-integrin, for example, by mechanically stretching cellsattached to ECM-coated flexible substrates (6). However, the molecularmechanism by which integrins mediate these ‘early-immediate’ mechanicalsignaling responses that activate TRPV4 are not well understood.

It is possible that the distal most region of the β1-integrincytoplasmic domain that contains a binding site for transmembrane aminoacid transporter CD98 mediates this near instantaneous integrindependent force induced calcium signal using magnetic pulling cytometry(MPC) and integrin chimera DNA constructions (7). CD98 is known toassociate with the β1-integrin cytoplasmic tail (8) and to be requiredfor adhesion strengthening through integrins as well as cytoskeletaltension-dependent fibronectin fibrillogenesis (9). However, themolecular mechanism whereby CD98 could play a role in the molecularconnection between these proteins is unclear. Described herein is dataindicating that CD98 mediates mechanical activation of the TRPV4membrane ion channel by physical forces applied to β1-integrins. CD98binds to both β1-integrin and TRPV4, and is required for mechanical, butnot chemical, activation of TRPV4.

Results

TRPV4 associates with CD98 and β1-integrin at focal adhesion. Theterminal 6 amino acid residues at the carboxy end of the β1-integrincytoplasmic mediate calcium signaling and are required for rapid forceinduced TRPV4-mediated calcium signaling (10). It was postulated thatCD98, which binds to the last 6 carboxy terminal amino acids ofβ1-integrin, is involved in the TRPV4 activation by β1-integrin usinggenetically engineered mutant integrin constructs. First, it wasconfirmed that CD98 binds to integrins through the region of theβ1-integrin tail using β1-integrin mutant constructs to transfectHEK293T cells followed by an immunoprecipitation assay. It was alsoconfirmed that TRPV4 also interacts with the same region of β1-integrinusing the same β1-integrin mutant constructs (data not shown). Toexplore the binding between those molecules, whether CD98 andβ1-integrin can be co-precipitated from HUVE cell lysates usinganti-TRPV4 antibody was determined (FIG. 1A). TRPV4 similarlyprecipitates with either anti-β1-integrin or anti-CD98 antibody (FIG.1B). Moreover, it was confirmed that TRPV4 overlapped with CD98 atvinculin positive focal adhesions by Total Internal ReflectionFluorescence (TIRF, data not shown). These findings suggest that thesethree molecules are structurally linked inside the focal adhesion at thecell's ECM binding site.

The deletion of the transmembrane region and C-terminal region of TRPV4did not affect the binding to either β1-integrin or CD98 (data notshown). TRPV4 has one proline rich domain and three ankyrin repeatdomains in its N-terminal region (FIG. 2A). Although TRPV4 lacking oneproline rich domain could associate with CD98 and β1-integrin, TRPV4lacking all three ankyrin repeat domains could not (FIG. 2B). This dataindicates that TRPV4 associates with β1-integrin and CD98 through itsankyrin repeat regions.

And also, the deletion of the C-terminal region of CD98 did no affectthe binding to either β1-integrin or TRPV4 (data not shown). In theN-terminal region of CD98, there is a region which was highly conservedfrom Drosophila to mammals (FIGS. 2C and 9). The deletion of this domainremoved the ability of CD98 to associate with either β1-integrin orTRPV4 (FIG. 2D). Thus, TRPV4, CD98 and β1-integrin make a complexthrough ankyrin repeat domain of TRPV4 and high homology domain of CD98.

CD98 mediates the binding between TRPV4 and β1-integrin at focaladhesion. The question is what the molecular mechanism is by which CD98mediates the signaling from β1-integrin to TRPV4. CD98 is important forsurface expression and clustering of β1-integrin in MCF7 cells (11).Therefore, it was examined whether CD98 mediates the binding betweenβ1-integrin and TRPV4 using siRNA-based knockdown of CD98. Transfectionof HUVE cells with CD98 siRNA resulted in decreased association of TRPV4and β1-integrin as well as loss of TRPV4 from focal adhesions that stillretained integrin and vinculin (FIG. 3). In addition, when monolayerCD98 siRNA-treated HUVE cells are scratched and cultured, the scratch isnormally restored by cell migration same as the cells treated withcontrol siRNA (data not shown). Thus, CD98 deficiency doesn't affect theformation of focal adhesion.

As described above, high homology domain of CD98 is important for thebinding to either β1-integrin or TRPV4. The effects of loss of only thatregion on β1-integrin-TRPV4 interaction were therefore examined.β1-integrin binding to TRPV4 was indeed inhibited by loss of the highhomology domain (FIG. 4). Therefore, CD98 mediates the binding betweenβ1-integrin and TRPV4 at focal adhesion through the high homology domainof CD98.

CD98 involves actin rearrangement on mechanotransduction. To confirmthat CD98 translates the mechanical signal from β1-integrin to TRPV4 bymediating the binding, it was examined whether CD98 is required for theactin arrangement induced by cyclic strain. To analyze it, HUVE cellswere cultured on flexible fibronectin-coated substrates and subjected to20% uniaxial cyclic strain using a FlexrCell™ Tension Plus system.Fluorescence microscope analysis of cells labeled with Alexa488-phalloidin combined with computerized morphometry revealed thatstress fibers thickened in these cells, and most realigned perpendicularto the main axis of the applied strain with 2 hours after forceapplication in control siRNA transfected HUVE cells. In contrast, thisresponse was abolished by the treatment with CD98 siRNA (FIG. 5).

Specific inhibition of mechanical activation of TRPV4 activity.Moreover, TRPV4 is specifically activated by mechanical strain (12). Toexamine whether CD98 transducse the signal by mechanical stimulationthrough TRPV4, the calcium influx by mechanical stimulation wasinvestigated. Transient increases in intracellular calcium were detectedquickly after force application in HUVE cells treated with controlsiRNA. However, HUVE cells treated with CD98 siRNA didn't show thisresponse (FIG. 6A). TRPV4 is also activated by chemical stimulation suchas 4α-phorbol 12, 13-didecanoate (4α-PDD). 4α-PDD produces a rapidincrease in intracellular calcium in HUVE cells treated with eithercontrol or CD98 siRNA (FIG. 6B), indicating that formation of theCD98-TRPV4-B1-Integrin complex is required for mechanical activation ofcalcium influx through TRPV4, but not for TRPV4 activation by chemicalstimuli.

Discussion

TRPV4 and β1-integrin are mechanosensors. The data presented hereinindicate that e that CD98 mediates the binding of TRPV4 to β1-integrins,and that it is required for mechanical, but not chemical, activation ofTRPV4. This study has revealed that the signaling pathways of TRPV4activation are different between mechanical and chemical cues.

TRPV4 has been implicated in the development of pulmonary edema inducedby various stimuli, including heart failure that acts by increasingpulmonary vascular pressure, as well as chemicals and inflammation.Importantly, in recent studies, pulmonary edema induced by IL2 wasstudied in a microengineered human “Lung-on-a-chip” microfluidic devicecontaining an artificial alveolar-capillary interface lined by livinghuman lung alveolar and capillary cells that experiences physiologicalbreathing motions and regenerates a functional vascular permeabilitybarrier in vitro (Huh D et al., 2010). Using this engineering approach,it was demonstrated that: a) IL2 produces clinical manifestations ofpulmonary edema (vascular leakage, fibrin clot formation, andcompromised oxygen transport) in vitro, b) physiological breathingmotions enhance IL2-induced increases in pulmonary vascular leakage by˜4-fold, and c) a chemical inhibitor of TRPV4 activity (e.g. GSK2193874)can prevent pulmonary vascular leakage and lung edema development in ahuman lung-on-a-chip microdevice that effectively mimics lungmicroarchitecture and physiology of the human alveolar-capillaryinterface (Huh D et al., 2012). Importantly, another group also hasshown that same TRPV4 inibitor prevents cardiogenic pulmonary edemainduced by increases pulmonary vascular pressure in rats, mice and dog(Thorneloe KS et al., 2012). These finding indicate that treatmentstargeting TRPV4 can permit new pulmonary edema therapies, and hence,understanding the molecular determinants responsible for mechanicalsignaling through TRPV4 has important clinical significance fortreatment of pulmonary edema.

TRPV4 is also present on many cell types and in many tissues, and it canbe activated by chemical (e.g., arachidonic acid metabolitesepoxyeicosatrienoic acids 5,6-EET and 8,9-EET) as well as mechanicalcues (e.g., breathing motions, ECM strain, fluid shear stress, pulmonaryvascular pressure, barotrauma). TRPV4 is also involved in the regulationof diverse bodily functions including control serum osmolarity,nociception and themal sensing and regulation in the central nervoussystem, bone formation and remodeling, bladder tone, motor and sensoryneuritogenesis, as well as inflammation and energy homeostasis inadipocytes. Thus, the breadth of functions of this ubiquitous membraneion channel would predict that systemic administration of inhibitorsTRPV4 activity might lead to diverse adverse effects and severe systemicdose-limiting toxicities. Thus, the identification of sites within CD98and TRPV4 that are responsible for their intermolecular bindinginteractions, as well as for mechanical activation of TRPV4 by physicalforces applied to integrins, permits the use more specific and/ortargeted inhibitors of pulmonary edema development.

Materials and Methods

Cell culture. Human Umbilical Vein Endothelial Cells (HUVE cells) andHuman embryonic kidney 293T (HEK293T) cells were obtained from theculture collection organization AMERICAN TYPE CULTURE COLLECTION™(ATCC). HUVE cells were cultured in supplemented endothelial growthmedium (EGM-2; Lonza). HEK293T cells were cultured in Dulbecco'smodified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum(FBS) and 100 units/ml penicillin and 100 μg/ml streptomycin.

siRNA knock down of CD98. RNA interference assay was conducted withhuman CD98 siRNA duplexes: 5′-GAAUGGUCUGGUGAAGAUC-3′ (SEQ ID NO: 21) and5′-GAUCUUCACCAGACCAUUC-3′ (SEQ ID NO: 22). Cells were transfected with50 nM of the siRNA duplexes in Opti-MEM™ (Invitrogen) using silentFectLipid Reagent for RNAi (BIO-RAD), according to the manufacturer'sinstructions.

Antibodies. Primary antibodies used for immunofluorescent staining,immunoprecipitation assay and immunoblotting included rabbit anti-ratTRPV4 antibody (Almone), mouse anti-human β1-integrin (BD TransductionLaboratories), rabbit anti-human CD98 antibody (abcam), mouseanti-chicken vinculin antibody (sigma) and Alexa fluor 488 phalloidin(Invitrogen).

Immunoprecipitation. Cells were extracted in ice-cold TRITON™ buffer (50mM Tris-HCL, pH 7.4, containing 150 mM NaCl, 1% TTRITONX100™, 5 mMEDTA). Cell extracts were centrifuged at 10000g for 15 min at 4° C.prior to being immunoprecipitated with each antibodies and DYNABEADS™Magnetic Beads (Invitrogen) at 4° C. Subsequently the beads wereisolated by magnet and washed five times with TRITON™ buffer and thenwere detected by SDS-PAGE and immunoblotting.

Mechanical strain application, measurement of cell orientation, CalciumImaging were performed as described (Thodeti et al., 2009)

References

TRPV4 channels mediate cyclic strain-induced endothelial cellreorientation through integrin-to-integrin signaling. Thodeti C K,Matthews B D, Ravi A, Mammoto A, Ghosh K, Bracha A L, Ingber DE. CircRes. 2009 104:1123-1130.

Ultra-rapid activation of TRPV4 ion channeks by mechanical forcesapplied to cell surface β1-integrins. Matthews B D, Thodeti C K, TytellJ D, Mammoto A, Overby D R, Ingber D E. Integr Biol. 2010 2:435-442

A human disease model of drug toxicity-induced pulmonary edema in alung-on-a-chip microdevice. Huh D, Leslie D C, Matthews B D, Fraser J P,Jurek S, Hamilton G A, Thorneloe K S, McAlexander M A, Ingber D E. SciTransl Med. 2012 4:159ra147

CD98 mediates mechanical signal from β1-integrin to TRPV4.Hirano-Kobayashi M, Matthews B D, Ingber D E. In preparation

Example 2 CD98 Mediates Mechanical Signaling from β1-integrin to TRPV4Channels in the Focal Adhesion

Cellular mechanotransduction—the process by which cells convert physicalforces applied on their surface into changes in intracellularbiochemistry and gene expression—is fundamental to development,physiology and disease. One of the most rapid (<5 msec)mechanotransduction events in human cells involves integrin-dependentactivation of the stress-sensitive membrane ion channel, TRPV4(Transient receptor potential vanilloid 4). However, the molecularmechanism by which force applied to cell surface integrins triggers this“early-immediate” mechanical signaling response is unknown, and itremains unclear whether this pathway is distinct from chemicalactivation mechanisms used by the channel It is described herein thatphysical forces applied to cell surface β1-integrins are transmitted tothe integrin-associated transmembrane protein CD98 that, in turn,directly transfers force to associated TRPV4 membrane ion channels,which results in their activation. B1-integrin, TRPV4 and CD98 allco-localize in focal adhesions in living cells, and they co-associatewithin common immunoprecipitable protein complexes. Recruitment of TRPV4can be prevented by suppressing CD98 expression with specific siRNA, andCD98 knock down also inhibits calcium influx through TRPV4 channelsinduced by mechanical forces, but not by chemical activators. Theportions of the β1-integrin, CD98 and TRPV4 molecules that mediate theseinteractions also have been identified. Together, these findingsdemonstrate that CD98 mediates rapid, mechanical force-inducedactivation of TRPV4 by facilitating direct force transfer betweenβ1-integrin and this ion channel within a common binding complex in thefocal adhesion.

Cellular mechanotransduction is critical for control of the growth anddevelopment of all tissues, and deregulation of this process contributesto the development of numerous diseases (ref1). Force sensing ismediated by cell surface integrin receptors (ref2), which is thought toinduce mechanochemical transduction by transmitting these stressesacross the cell surface and inducing deformations in proteins, such astalin and vinculin (ref3), that form the cytoskeletal backbone of thefocal adhesion. However, the finding that forces application toβ1-integrins results in almost immediate (<5 millisec) activation ofcalcium influx through TRPV4 membrane ion channels in endothelial cells(ref4) raises the possibility that ultra-rapid mechanochemicalconversion is mediated by direct mechanical signal transfer fromintegrins to TRPV4 channels. Understanding this mechanism is importantbecause mechanical activation of TRPV4 channels in endothelial cells hasbeen shown to play a central role in the development of pulmonary edema,and TRPV4 antagonists can effectively suppress development of thesedisease (refs, 6). Thus, described herein is the exploration of howmechanical forces applied to integrins activate TRPV4 channels on theendothelial cell surface.

It was first examined whether the transmembrane amino acid transporterCD98 mediates force transfer between β1-integrin and TRPV4. When HEK293Tcells were transfected with β1-integrin mutant constructs andimmunoprecipitation assays performed, it was confirmed that CD98specifically binds to the β1-integrin tail region (FIG. 8B).Interesting, using the same approach, it was found that TRPV4 alsointeracts with the same region of β1-integrin molecule (FIG. 8B). Inseparate studies with human umbilical vein endothelial (HUVE) cells, itwas found that CD98 and β1-integrin can be co-precipitated usinganti-TRPV4 antibody (FIG. 1A), and that TRPV4 can be similarlyprecipitated using either anti-β1-integrin or anti-CD98 antibodies (FIG.1B). Moreover, TRPV4 colocalizes with both CD98 and vinculin withinspontaneously formed focal adhesions, when analyzed by total internalreflection fluorescence (TIRF) microscopy (data not shown). Thesefindings indicated that β1-integrin, CD98 and TRPV4 co-associate witheach other and with cytoskeletal proteins within focal adhesions.

To explore in greater detail how TRPV4 senses mechanical signals, thetransmembrane and C-terminal regions of TRPV4 were genetically deleted;however, this did not alter its ability to bind to either β1-integrin orCD98 when analyzed using the immunoprecipitation assay (FIGS. 2A-2D).Similarly, when the proline rich domain of TRPV4 was deleted, it alsohad no effect on its ability to associate with these two molecules (FIG.2B). However, deletion of the three ankyrin repeat domains (237-266aa,284-313aa, and 369-398aa) in the N-terminus of TRPV4, was sufficient tocompletely inhibit binding to both β1-integrin and CD98 (FIG. 2B).

Thus, the findings suggest that the ankyrin repeat regions of TRPV4mediate formation of the integrin-CD98-TRPV4 complex; however, it doesnot clarify how CD98 is recruited to this complex. CD98 is also amembrane protein composed of an N-terminal cytoplasmic domain, anintervening transmembrane domain, and C-terminal extracellular domain.Deletion of the C-terminal region of CD98 did not alter its ability tobind to either β1-integrin or TRPV4 (FIG. 2D). In contrast, deletion ofa 50 amino acid (160-210aa) high homology domain within the CD98N-terminus that is highly conserved from drosophila to mammalscompletely blocked its ability to associate with either β1-integrin orTRPV4 (FIG. 9). Thus, β1-integrin, CD98 and TRPV4 co-associate throughbinding interactions between the last 6aa residues on the integrincytoplasm tail, the high homology domain of CD98 and the ankyrin repeatdomains of TRPV4.

It was next asked whether CD98 serves to link β1-integrin to TRPV4.Knock down of CD98 expression in HUVE cells using siRNA greatly reducedthe amount of both CD98 and TRPV4 within focal adhesions, although ithad no effect on recruitment of either integrins or vinculin (data notshown) to these anchoring complexes, and it did not interfere with cellmigration in scratch wound assays (data not shown). Thus, CD98 is notrequired for focal adhesion formation or function. However knock down ofCD98 expression in HUVE cells using siRNA greatly reduced the amount ofthe binding between β1-integrin and TRPV4 (FIG. 3). Although TRPV4expression was not decreased, TRPV4 couldn't localize at focal adhesions(data not shown).

As described above, high homology domain of CD98 is important for thebinging to either β1-integrin or TRPV4. It was next examined if the highhomology domain of CD98 shows dominant negative effect byimmunoprecipitation assay and stretching assay. β1-integrin binding toTRPV4 was decreased by overexpressing high homology domain of CD98 (FIG.4). And the cells overexpressing high homology domain of CD98 didn'tshow actin rearrangements after giving mechanical strain (FIG. 12).These data indicate that the high homology domain of CD98 can competewith endogenous CD98 on mediating the binding between TRPV4 andβ1-integrin. Therefore, CD98 mediates the binding between β1-integrinand TRPV4 at focal adhesion through high homology domain of CD98.

CD98 involves actin rearrangement on mechanotransduction

To confirm that CD98 translates the mechanical signal from β1-integrinto TRPV4 by mediating the binding, it was examined whether CD98 isrequired for the actin arrangement induced by cyclic strain. To analyzeit, HUVE cells were cultured on flexible fibronectin-coated substratesand subjected to 20% uniaxial cyclic strain using a FlexrCell TensionPlus™ system. Fluorescence microscope analysis of cells labeled withAlexa 488-phalloidin combined with computerized morphology revealed thatstress fibers thickened in these cells, and most realigned perpendicularto the main axis of the applied strain with 2 hours after forceapplication in control siRNA transfected HUVE cells. In contrast, thisresponse was abolished by the treatment with CD98 siRNA (FIG. 5).

Moreover, TRPV4 is specifically activated by mechanical strain (ref10).To examine whether CD98 transduce the signal by mechanical stimulationthrough TRPV4, the calcium influx by mechanical stimulation wasexamined. Transient increases in intracellular calcium were detectedquickly after force application in HUVE cells treated with controlsiRNA. However, HUVE cells treated with CD98 siRNA didn't show thisresponse (FIG. 6A). TRPV4 is also activated by chemical stimulation suchas 4a-phorbol 12, 13-didecanoate (4a-PDD). 4a-PDD produces a rapidincrease in intracellular calcium in HUVE cells treated with eithercontrol or CD98 siRNA (FIG. 6B), indicating that CD98 plays a role inthe calcium influx through the activation of TRPV4 by mechanical, butnot chemical.

TRPV4 and β1-integrin are mechanosensors.

The molecular mechanism between mechanosensors is still unclear. It isproposed herein that CD98 mediates the binding of TRPV4 to β1-integrins,and that it is required for mechanical, but not chemical, activation ofTRPV4. This study has revealed that the signaling pathway of TRPV4activation is different between mechanical and chemical cue.

Surprisingly, CD98 mediated only mechanical cues from β1-integrin toTRPV4. Although the localization of TRPV4 at focal adhesion wasdecreased in CD98 knockdown cells, TRPV4 localization was visible at thecell membrane by TIRF. These data indicate that CD98 mediates TRPV4 tolocalize at focal adhesion and transfers mechanical cues fromβ1-integrin.

TRPV4 is phosphorylated when it is activated (ref8). The cytoplasmicregion of β1-integrin interacts with many kinases such as FAK, ILK andSrc. Without wishing to be limited by theory, it is contemplated hereinthat a certain kinase, which is included in integrin complex,phosphorylates TRPV4 in response to mechanical cues.

TRPV4 has been implicated in the development of pulmonary edema inducedby various stimuli, including heart failure that acts by increasingpulmonary vascular pressure, as well as chemicals and inflammation.Importantly, pulmonary edema induced by IL2 has been studied in amicroengineered human “Lung-on-a-chip” microfluidic device containing anartificial alveolar-capillary interface lined by living human lungalveolar and capillary cells that experiences physiological breathingmotions and regenerates a functional vascular permeability barrier invitro (ref9). Using this engineering approach, it was demonstrated that:a) IL2 produces clinical manifestations of pulmonary edema (vascularleakage, fibrin clot formation, and compromised oxygen transport) invitro, b) physiological breathing motions enhance IL2-induced increasesin pulmonary vascular leakage by ˜4-fold, and c) a chemical inhibitor ofTRPV4 activity (GSK2193874) can prevent pulmonary vascular leakage andlung edema development in a human lung-on-a-chip microdevice thateffectively mimics lung microarchitecture and physiology of the humanalveolar-capillary interface (ref11). That same TRPV4 inhibitor preventscardiogenic pulmonary edema induced by increasing pulmonary vascularpressure in rats, mice and dog (ref12).

Thus, TRPV4 can be a novel target for new pulmonary edema therapies, andhence, understanding the molecular determinants responsible formechanical signaling through TRPV4 can have important clinicalsignificance for treatment of pulmonary edema. But TRPV4 is also presenton many cell types and in many tissues, and it can be activated bychemical (e.g., arachidonic acid metabolites epoxyeicosatrienoic acids5,6-EET and 8,9-EET) as well as mechanical cues (e.g., breathingmotions, ECM strain, fluid shear stress, pulmonary vascular pressure,barotrauma). TRPV4 is also involved in the regulation of diverse bodilyfunctions including control serum osmolality, nociception and thermalsensing and regulation in the central nervous system, bone formation andremodeling, bladder tone, motor and sensory neuritogenesis, as well asinflammation and energy homeostasis in adipocytes (ref18-37). Thus, thebreadth of functions of this ubiquitous membrane ion channel predictthat systemic administration of inhibitors of TRPV4 activity can lead todiverse adverse effects and severe systemic dose-limiting toxicities.Thus, the identification of sites within CD98 and TRPV4 that areresponsible for their intermolecular binding interactions, as well asfor mechanical activation of TRPV4 by physical forces applied tointegrins, can lead to the development of new and improved inhibitors ofpulmonary edema development. Described herein are methods andcompositions that permit specific inhibition of TPRV4 activation due tomechanical cues.

Methods

Methods were as detailed in Example 1

REFERENCES

-   1. Ingber D E. Mechanobiology and diseases of mechanotransduction.    Ann Med 2003;35:564-77.-   2. Wang N, Butler J P and Ingber D E. Mechanotransduction across the    cell surface and through the cytoskeleon. Science 1993-   3. Wirtz H R, Dobbs L G. The effects of mechanical forces on lung    functions. Respir Physiol. 2000;119:1-17.-   4. Matthews B D, Thodeti C K, Tytell J D, Mammoto A, Overby D R,    Ingber D E. Ultra-rapid activation of TRPV4 ion channels by    mechanical forces applied to cell surface betal integrins. Integr    Biol (Camb) 2010;2:435-42.-   5. Thorneloe K S, Cheung M, Bao W, et al. An orally active TRPV4    channel blocker prevents and resolves pulmonary edema induced by    heart failure. Sci Transl Med 2012;4:159ra48.-   6. Huh D, Leslie D C, Matthews B D, et al. A human disease model of    drug toxicity-induced pulmonary edema in a lung-on-a-chip    microdevice. Sci Transl Med 2012;4:159ra47.-   7. Thodeti C K, Matthews B, Ravi A, et al. TRPV4 channels mediate    cyclic strain-induced endothelial cell reorientation through    integrin-to-integrin signaling. Circ Res 2009;104:1123-30.-   8. Kolesnikova T V, Mannion B A, Berditchevski F, Hemler M E. Betal    integrins show specific association with CD98 protein in low density    membranes. BMC Biochem 2001;2:10-   9. Feral C C, Zijlstra A, Tkachenko E, Prager G, Gardel M L, Slepak    M, Ginsberg M H. CD98 hc (SLC3A2) participates in fibronectin matrix    assembly by mediating integrin signaling. J Cell Biol.    2007;178:701-11.-   10. Mammoto A, Mammoto T, Ingber D E. Mechanosensitive mechanisms in    transcriptional regulation. J Cell Sci 2012;125:3061-73.-   11. Cai S, Bulus N, Fonseca-Siesser P M, Chen D, Hanks S K, Pozzi A,    Zent R. CD98 modulates integrin betal function in polarized    epithelial cells. J Cell Sci. 2005;118:889-99.-   12. Adapala R K, Talasila P K, Bratz I N, Zhang D X, Suzuki M,    Meszaros J G, Thodeti C K. PKCα mediates acetylcholine-induced    activation of TRPV4-dependent calcium influx in endothelial cells.    Am J Physiol Heart Circ Physiol 2011;301:H757-65.-   13. Thorneloe K S, Cheung M, Bao W, et al. An Orally Active TRPV4    Channel Blocker Prevents and Resolves Pulmonary Edema Induced by    Heart Failure. . Sci Transl Med 2012;4:159ra148-   14. Liedtke W. TRPV4 as osmosensor: a transgenic approach. Pflugers    Arch 2005;451:176-80.-   15. Liedtke W, Friedman JM. Abnormal osmotic regulation in trpv4−/−    mice. Proc Natl Acad Sci USA 2003;100:13698-703.-   16. Strotmann R, Harteneck C, Nunnenmacher K, Schultz G, Plant T D.    OTRPC4, a nonselective cation channel that confers sensitivity to    extracellular osmolarity. Nat Cell Biol 2000;2:695-702.-   17. Liedtke W, Choe Y, Marti-Renom M A, et al. Vanilloid    receptor-related osmotically activated channel (VR-OAC), a candidate    vertebrate osmoreceptor. Cell 2000;103:525-35.-   18. Mizuno A, Matsumoto N, Imai M, Suzuki M. Impaired osmotic    sensation in mice lacking TRPV4. Am J Physiol Cell Physiol    2003;285:C96-101.-   19. Jin M, Berrout J, Chen L, O'Neil R G. Hypotonicity-induced TRPV4    function in renal collecting duct cells: modulation by progressive    cross-talk with Ca2+-activated K+channels. Cell calcium    2012;51:131-9.-   20. Chen L, Liu C, Liu L. Osmolality-induced tuning of action    potentials in trigeminal ganglion neurons. Neuroscience letters    2009;452:79-83.-   21. Li J, Wang M H, Wang L, et al. Role of transient receptor    potential vanilloid 4 in the effect of osmotic pressure on    myocardial contractility in rat. Sheng li xue bao : [Acta    physiologica Sinica] 2008;60:181-8.-   22. Zhang Y, Wang Y H, Ge H Y, Arendt-Nielsen L, Wang R, Yue S W. A    transient receptor potential vanilloid 4 contributes to mechanical    allodynia following chronic compression of dorsal root ganglion in    rats. Neuroscience letters 2008;432:222-7.-   23. Alessandri-Haber N, Yeh J J, Boyd A E, et al. Hypotonicity    induces TRPV4-mediated nociception in rat. Neuron 2003;39:497-511.-   24. Bang S, Yang T J, Yoo S, Heo T H, Hwang S W. Inhibition of    sensory neuronal TRPs contributes to anti-nociception by butamben.    Neuroscience letters 2012;506:297-302.-   25. Liu T T, Bi H S, Lv S Y, Wang X R, Yue S W. Inhibition of the    expression and function of TRPV4 by RNA interference in dorsal root    ganglion. Neurological research 2010;32:466-71.-   26. Guler A D, Lee H, lida T, Shimizu I, Tominaga M, Caterina M.    Heat-evoked activation of the ion channel, TRPV4. J Neurosci    2002;22:6408-14.-   27. Chung M K, Lee H, Caterina M J. Warm temperatures activate TRPV4    in mouse 308 keratinocytes. J Biol Chem 2003;278:32037-46.-   28. Mizoguchi F, Mizuno A, Hayata T, et al. Transient receptor    potential vanilloid 4 deficiency suppresses unloading-induced bone    loss. Journal of cellular physiology 2008;216:47-53.-   29. Rock M J, Prenen J, Funari V A, et al. Gain-of-function    mutations in TRPV4 cause autosomal dominant brachyolmia. Nat Genet    2008;40:999-1003.-   30. Krakow D, Vriens J, Camacho N, et al. Mutations in the gene    encoding the calcium-permeable ion channel TRPV4 produce    spondylometaphyseal dysplasia, Kozlowski type and metatropic    dysplasia. American journal of human genetics 2009;84:307-15.-   31. Birder L, Kullmann F A, Lee H, et al. Activation of urothelial    transient receptor potential vanilloid 4 by 4alpha-phorbol    12,13-didecanoate contributes to altered bladder reflexes in the    rat. J Pharmacol Exp Ther 2007;323:227-35.-   32. Gevaert T, Vriens J, Segal A, et al. Deletion of the transient    receptor potential cation channel TRPV4 impairs murine bladder    voiding. J Clin Invest 2007;117:3453-62.-   33. Thorneloe K S, Sulpizio A C, Lin Z, et al.    N-((1S)-1-{[4-((2S)-2-{[(2,4-dichlorophenyl)sulfonyl]amino}-3-hydroxypropanoyl)-1-piperazinyl]carbonyl}-3-methylbutyl)-1-benzothiophene-2-carboxamide    (GSK1016790A), a novel and potent transient receptor potential    vanilloid 4 channel agonist induces urinary bladder contraction and    hyperactivity: Part I. J Pharmacol Exp Ther 2008;326:432-42.-   34. Xu X, Gordon E, Lin Z, Lozinskaya I M, Chen Y, Thorneloe K S.    Functional TRPV4 channels and an absence of capsaicin-evoked    currents in freshly-isolated, guinea-pig urothelial cells Channels    (Austin) 2009;3:156-60.-   35. Jang Y, Jung J, Kim H, et al. Axonal neuropathy-associated TRPV4    regulates neurotrophic factor-derived axonal growth. J Biol Chem    2012;287:6014-24.-   36. Ye L, Kleiner S, Wu J, et al. TRPV4 Is a Regulator of Adipose    Oxidative Metabolism, Inflammation, and Energy Homeostasis. Cell    2012;151:96-110.-   37. Roca-Cusachs P, Iskratsch T, Sheetz M P. Finding the weakest    link - exploring integrin-mediated mechanical molecular pathways. J    Cell Sci 2012;125:3025-38.-   38. Thodeti C K, Matthews B, Ravi A, et al. TRPV4 channels mediate    cyclic strain-induced endothelial cell reorientation through    integrin-to-integrin signaling. Circ Res 2009;104:1123-30.

What is claimed herein is:
 1. A method of inhibiting themechanically-dependent activation of TRPV4, the method comprisingcontacting a cell with a compound comprising a polypeptide comprising asequence with at least 95% identity to the sequence of SEQ ID NO: 1, ora nucleic acid encoding said polypeptide.
 2. The method of claim 1,wherein polypeptide comprising the sequence of SEQ ID NO:
 1. 3. Themethod of claim 1, wherein polypeptide comprising a sequence with atleast 95% identity to the sequence of SEQ ID NO: 1 comprises no morethan 100 amino acids.
 4. The method of claim 1, wherein the polypeptidecomprising a sequence with at least 95% identity to the sequence of SEQID NO: 1 is capable of inhibiting the binding of Transient ReceptorPotential Vanilloid 4 (TRPV4) or integrin to Cluster of Differentiation98 (CD98).
 5. The method of claim 1, wherein the nucleic acid is a cDNA.6. The method of claim 1, wherein the nucleic acid is in the form of avector comprising the nucleic acid.
 7. The method of claim 1, whereinthe nucleic acid is in the form of a viral vector comprising the nucleicacid.
 8. The method of claim 7, wherein the vector is anadeno-associated virus (AAV) vector.
 9. The method of claim 1, whereinthe compound further comprises a cell-penetrating agent.
 10. The methodof claim 9, wherein the cell-penetrating agent is TAT polypeptide orBioporter.
 11. The method of claim 1, wherein the compound is caused topenetrate a cell via electroporation or magnetoporation.
 12. A method oftreating a disease in a subject, the method comprising administering acompound comprising a polypeptide comprising a sequence with at least95% identity to the sequence of SEQ ID NO: 1, or a nucleic acid encodingsaid polypeptide to the subject
 13. The method of claim 12, wherein thedisease is selected from the group consisting of: pulmonary edema;systemic edema; hypertension; hyperalgesia; inflammation; brachyolmia;spondylometaphyseal dysplasia Kozlowski type; metatropic dysplasia;peripheral neuropathy; asthma; COPD; overactive bladder; incontinence;and acoustic cochlear injury.
 14. The method of claim 12, whereinpolypeptide comprising the sequence of SEQ ID NO:
 1. 15. The method ofclaim 12, wherein polypeptide comprising a sequence with at least 95%identity to the sequence of SEQ ID NO: 1 comprises no more than 100amino acids.
 16. The method of claim 12, wherein the polypeptidecomprising a sequence with at least 95% identity to the sequence of SEQID NO: 1 is capable of inhibiting the binding of Transient ReceptorPotential Vanilloid 4 (TRPV4) or integrin to Cluster of Differentiation98 (CD98).
 17. The method of claim 1, wherein the nucleic acid is acDNA.
 18. The method of claim 1, wherein the nucleic acid is in the formof a vector, viral vector, or adeno-associated virus (AAV) vectorcomprising the nucleic acid.
 19. The method of claim 1, wherein thecompound further comprises a cell-penetrating agent.
 20. A method ofidentifying an inhibitor of the mechanically-dependent activation ofTRPV4, the method comprising: contacting a complex comprising TRPV4 andCD98, and optionally, integrin with a candidate agent; measuring thelevel of complexed TRPV4 and CD98, and optionally, integrin; wherein adecrease in the level of TRPV4 complexed with CD98, or the level ofintegrin complexed with either TRPV or CD98 indicates the candidate caninhibit the mechanically-dependent activation of TRPV4.